Volume 5 Supplement 7

IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery

Open Access

Novel BAP degradation pathway during adventitious caulogenesis in Pinus pineaL. cotyledons

  • Ondrej Novak1Email author,
  • Candela Cuesta2,
  • Karel Dolezal1,
  • Lucie Szucova1,
  • Lukas Spichal1,
  • Belen Fernandez2,
  • Ana Rodriguez2 and
  • Miroslav Strnad1
BMC Proceedings20115(Suppl 7):P143

DOI: 10.1186/1753-6561-5-S7-P143

Published: 13 September 2011

Background

Cytokinins (CKs) are a group of phytohormones which probably regulate the growth, development, and metabolism of all plants. The aromatic CK benzyladenine (BA) has been widely applied in in vitro culture for inducing shoot organogenesis. Our study of endogenous cytokinin profiles during the caulogenic process based on mature cotyledons of stone pine (Pinus pinea L.) showed a novel metabolic pathway of aromatic cytokinins based on modification of purine skeleton.

Methods

Three-year-old mature seeds from two half-sibling selected families and open-pollinated trees of P. pinea were used [1] and the samples were collected following the Alonso et al.[2] procedure. Extraction and purification of cytokinins was based on the method described by Novák et al. [3], including modifications published later [4]. The samples were purified using a combination of a cation (SCX-cartridge) and anion [DEAE-Sephadex/C18-cartridge] exchangers. Combination of high performance liquid chromatography (HPLC) with quadrupole-time of flight mass spectrometry (QqTOF) was used for accurate and sensitive identification and quantification of cytokinins.

Results and discussion

Cortizo et al. [5] published the dynamics of BA uptake and metabolism in P. pinea cotyledons excised from embryos precultured for 2 and 4 days and cultured in vitro in modified Le Poivre media with 4.4 µM BA. In our experiment, we used 44.4 µM concentration of BA and samples were collected at different periods (0; 1; 2; 6; 16; 24 h and 2; 4; 6 d). Using high-resolution MS, the naturally-occurring BA metabolites as well as new BA forms were identified. In comparison with previously published profiles of the BA metabolite pool [5, 6], the ribosyl and glycosyl forms were quantified as the most abundant metabolites. Moreover the biological activity of identified BA and its derivatives were compared in various CK bioassays. The results indicate that BA uptake during the caulogenic process may be possible to regulate not only by known cytokinin pathways. Finally, the feeding experiment with stable isotope-labelled standard, 15N4-BA, confirmed our identification of the novel metabolic CK pathway.

Conclusions

The identification of the novel BA forms demonstrates that the novel cytokinin pathway is used as a control mechanism of BA uptake from the bud induction medium during adventitious caulogenesis in cotyledons of P. pinea. Our results help to understand the processes associated with embryo germination in plant tissue culture.

Authors’ Affiliations

(1)
Laboratory of Growth Regulators, Faculty of Science, Palacky University & Institute of Experimental Botany AS CR
(2)
Area de Fisiologia Vegetal, Departamento Biologia de Organismos y Sistemas, Instituto de Biotecnologia de Asturias, Universidad de Oviedo

References

  1. Cuesta C, Ordas RJ, Fernandez B, Rodriguez A: Clonal micropropagation of six selected half-sibling families of Pinus pinea and somaclonal variation analysis. Plant Cell Tiss. Org. 2008, 95: 125-130. 10.1007/s11240-008-9412-y.View ArticleGoogle Scholar
  2. Alonso P, Moncalean P, Fernandez B, Rodriguez A, Centeno ML, Ordas RJ: An improved micropropagation protocol for stone pine (Pinus pinea L.). Ann. Forest Sci. 2006, 63: 879-885. 10.1051/forest:2006071.View ArticleGoogle Scholar
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  4. Novak O, Hauserova E, Amakorova P, Dolezal K, Strnad M: Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Phytochem. 2008, 69: 2214-2224. 10.1016/j.phytochem.2008.04.022.View ArticleGoogle Scholar
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Copyright

© Novak et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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