BIOLOGIA PLANTARUM 57 (1): 174-178, 2013 DOI:10.1007/s10535-012-0252-6 174 BRIEF COMMUNICATION The role of cytokinins during micropropagation of wych elm J. MALÁ 1 , P. MÁCHOVÁ 1 , H. CVRČKOVÁ 1 , M. KARADY 2 , O. NOVÁK 2 , J. MIKULÍK 2 , J. DOSTÁL 1 , M. STRNAD 2,3 , and K. DOLEŽAL 2,3 * Forestry and Game Management Research Institute, Strnady 136, CZ-25202 Jíloviště, Czech Republic 1 Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany AS CR, CZ-78371 Olomouc-Holice, Czech Republic 2 Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic 3 Abstract We have compared the influence of two aromatic cytokinin derivatives, N 6 -benzyladenine (BA) and meta-topolin (mT), on the in vitro multiplication and senescence of wych elm (Ulmus glabra Huds.). After 3 months of cultivation, the micropropagation rate was higher (approx. six times more shoots developed) on Murashige and Skoog (MS) medium supplemented with mT than on MS supplemented with BA. Quantification of 50 endogenous cytokinins, using a recently developed UPLC-ESI(+)-MS/MS method, showed significant differences in the cytokinin metabolites (especially different glucosides) in explants cultivated on media supplemented with BA or mT. Differences in efficiency of photosystem II was also detected. Additional key words: benzylaminopurine, chlorophyll fluorescence, meta-topolin, organogenesis, rooting, senescence, Ulmus glabra. ⎯⎯⎯⎯ Elms (Ulmus sp.) are trees that are highly valued for their great strength, tightly twisted grain, durability, and tolerance of both cold stress and salinity (Heybroek et al. 1982). Wych elm (Ulmus glabra Huds.) is a native European species that is common in mountainous regions of the Czech Republic. However, during the 1970’s, the disease epidemic destroyed most elm populations in Europe (Brasier 1990, Fenning et al. 1993). Hence, a program for conserving genetic resources of elms has been initiated, within the EUFORGEN framework, which includes establishment of collections of trees (clonal archives), presuming trees that survived the epidemic to have a high degree of resistance against repeated infection. Elite elm trees can be vegetatively propagated by grafting and cuttings but micropropagation is the most promising option (Gartland et al. 2004). However, although various micropropagation systems have been reported for numerous species and hybrids (Biondi et al. 1984, Fink et al. 1986, Chalupa 1994, Fenning et al. 1993, Corchete et al. 1993, Cheng and Shi 1995, Malá et al. 2005), some obstacles still remain during organo- genesis, notably early senescence and low multiplication rates, both of which decrease the micropropagation efficiency. Optimizing micropropagation is not straightforward since it is a complex process involving a sequence of developmental stages that are influenced by numerous endogenous and exogenous stimuli (Mohamed and Alsadon 2011). These stimuli include phytohormones, especially auxins and cytokinins (Pumisutapon et al. ⎯⎯⎯⎯ Received 29 July 2011, accepted 5 April 2012. Abbreviations: BA - N 6 -benzyladenine; cZ - cis-zeatin; DHZ - dihydrozeatin; F v /F m - variable to maximum chlorophyll fluorecence ratio; *9G - 9-β-D-glucopyranosyl derivative; IAC - immunoaffinity chromatography; IBA - indole-3-butyric acid; iP - N 6 -isopentenyladenine; *MP - 5´-monophosphate derivative; LC-MS - liquid chromatography combined with mass spectrometry; MS - Murashige and Skoog medium; mT - meta-topolin [6-(3-hydroxybenzylamino)purine]; *OG - O-β-D-gluco- pyranosyl derivative; oT - ortho-topolin; PS II - photosystem II; *R - 9-β-D-ribofuranosyl derivative; tZ - trans-zeatin; UPLC-ESI(+)-MS/MS - ultra performance liquid chromatography combined with positive electrospray mass spectrometry. Acknowledgements: This work was supported by grants from the Ministry of Agriculture of the Czech Republic (NAZV QI 92A247), Czech Ministry of Education (MSM 6198959216), and ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research. * Corresponding author; fax: (+420) 585634870, e-mail: karel.dolezal@upol.cz