biologia plantarum

Biologia plantarum 52:329-333, 2008 | DOI: 10.1007/s10535-008-0067-7

Cryopreservation of cell suspension cultures of Taxus × media and Taxus floridana

K. Škrlep¹, M. Bergant^1,2,5, G. M. De Winter², B. Bohanec³, J. Žel¹, R. Verpoorte⁴, F. Van Iren², M. Camloh^1,*

¹ National Institute of Biology, Ljubljana, Slovenia

² Institute of Biology, Leiden University, AL Leiden, The Netherlands

³ Biotechnical faculty, University of Ljubljana, Ljubljana, Slovenia

⁴ Institute of Biology, Leiden University, CC Leiden, The Netherlands

⁵ University of Nova Gorica, Nova Gorica, Slovenia

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately -1 °C per min to -80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.

Keywords: cryo-storage; flow cytometry; genetic stability; yew

Subjects: cryopreservation; genetic stability; in vitro culture, cell suspension; in vitro culture, growth control; Taxus floridana; yew

Received: November 25, 2005; Accepted: January 20, 2006; Published: June 1, 2008  Show citation