Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce)
2010
Hazubska-Przybył, Teresa | Chmielarz, Paweł | Michalak, Marcin | Bojarczuk, Krystyna
Two-year-old embryogenic tissues (ET) of Picea omorika (Pančić) Purk. were successfully cryopreserved after preculture with sucrose, air-drying for 2 h, and freezing in liquid nitrogen (LN). The preculture protocol consisted of passaging the ET onto standard Litvay medium containing increasing concentrations of sucrose (0.25 M sucrose for 24 h, 0.5 M for 24 h, 0.75 M for 2 days, and 1.0 M for 3 days) for 7 days, at 25°C, and in the dark. The clumps were subsequently air-dried over silica gel, down to a 20% water content (based on fresh weight), placed in cryovials, and immersed in liquid nitrogen (LN) for 24 h. These were thawed at 42°C and progressively rehydrated in phytagel-solidified LM media containing decreasing concentrations of sucrose. After 3 weeks of in vitro culture, surviving clumps were friable and white in color, similar to their morphology prior to cryostorage. The frequency of bacterial and fungal contamination was higher if ET was frozen in LN-containing vials than in LN-free vials. This efficient cryopreservation protocol would be useful for the ex-situ conservation of P. omorika germplasm in gene banks at very low temperatures.
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