DOI: 10.1007/s10535-013-0357-6 BIOLOGIA PLANTARUM 57 (4): 793-796, 2013 793 BRIEF COMMUNICATION Chloroplast ultrastructure of Hypericum perforatum plants regenerated in vitro after cryopreservation D. STOYANOVA-KOLEVA 1 *, M. STEFANOVA 1 , E. ČELLÁROVÁ 2 , and V. KAPCHINA-TOTEVA 3 Department of Botany 1 and Department of Plant Physiology 3 , Faculty of Biology, Sofia University “St. Kliment Ohridski”, BG-1408 Sofia, Bulgaria Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, SK-04180 Košice, Slovakia 2 Abstract The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min -1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min -1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation. Additional key words: ABA, mannitol, medicinal plant, micropropagation, St. John’s wort. ⎯⎯⎯⎯ Cryo-procedures are innovative technique which allows preservation of plant material with valuable qualities at ultra-low temperatures for theoretically unlimited period of time (Ozudogru et al. 2010). The key factor for successful cryopreservation is minimizing damages of the cell ultrastructure (Xu et al. 2006, Fki et al. 2013). Physiological, biochemical, and genetic properties of Hypericum perforatum L. plants regenerated after cryopreservation were tested previously (Urbanová et al. 2002, 2006, 2010, Skyba et al. 2010) but the structural organization of the plastid apparatus has not been thoroughly examined. According to some studies, a common reaction to low temperatures is a collapse of the photosynthetic apparatus (Danova et al. 2009, Ganeva et al. 2009). Studies of the leaf structure of in vitro cultured plants revealed that the examination on subcellular level is very important for evaluation of plant regeneration potential (Stefanova 2011, Stoyanova- Koleva et al. 2012). The aim of this study was to establish the chloroplast ultrastructure of H. perforatum plants propagated in vitro without and after cryopreservation and to analyze the alterations of some variables during precryogenic/cryoge- nic treatments. The obtained results would be very useful in elaborating the protocol for successful cryopreservation. The photosynthetic apparatus of in vitro cultured Hypericum perforatum L. plants regenerated after cryo- preservation was investigated by transmission electron microscopy (TEM). After 14 - 21 d of in vitro culture on standard Murashige and Skoog (1962; MS) medium containing vitamins according to Gamborg et al. (1968), 30 g dm -3 sucrose, 2 mg dm -3 glycine, and 100 mg dm -3 myo-inositol (pH 5.6), 140 isolated shoot tips were pretreated with 0.076 µM abscisic acid (ABA) for 10 d or 0.3 M mannitol for 7 d. Cryopreservation was performed according to Skyba et al. (2011). The shoot tips were subjected to controlled cooling at different rates: 0.3, 0.2, and 0.1 °C min -1 . Survival was determined as percentage of explants capable of regrowth and regeneration from the initial number of frozen tips. The cryopreserved shoot tips regenerated on the MS medium supplemented with 0.5 mg dm -3 benzyladenine (BA). Shoot tips which did not undergo cryo-procedure and regenerated on MS medium without growth regulators served as control plants. ⎯⎯⎯⎯ Received 21 January 2013, accepted 29 April 2013. Abbreviations: ABA - abscisic acid; BA - benzyladenine. Acknowledgements: This work was supported by grants from the Bulgarian Ministry of Education, Youth, and Science (Project DNTC BG-SK-01/2012) and from the Slovak Research and Development Agency (Project APVV SK-BG 0012-10). * Corresponding author; fax: (+359) 2 865 66 41, e-mail: koleva_phd@abv.bg