Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce)

Abstract

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.

A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer

“…In order to stimulate embryo development and maturation, osmoticum (sucrose or maltose) and phytagel of higher concentrations were supplemented in the medium. Lower water potential in culture medium can result in lower water content in plant tissue that benefit embryo maturation (Stasolla et al 2002) and also freezing tolerance (Yamazak et al 2009;Hazubska-Przybyl et al 2010). As non-plasmolyzing osmoticum, PEG enhances the maturation of spruce somatic embryos in both number and quality (Attree et al 1991;Kong and Yeung 1995;Kong et al 1998;.…”

Cryopreservation of in vitro-grown shoot-tips of Rabdosia rubescens by encapsulation-dehydration and evaluation of their genetic stability

“…In vitro tissue culture conservation is susceptible to contamination and somaclonal variation (Watt et al 2009;Gonçalves et al 2010). Cryopreservation is the most promising choice for conservation of vegetatively propagated plants owing to its safety, repeatability and long-term storage possibility (Pacheco et al 2009;Hazubska-Przbyl et al 2010). Cryogenic procedures such as simple-freezing, vitrification, encapsulation-dehydration and encapsulation-vitrification have been developed and the number of cryopreserved species or cultivars has sharply increased (Tsai et al 2009;Hua and Hong 2010).…”

Somaclonal Variation During Picea abies and P. omorika Somatic Embryogenesis and Cryopreservation

“…However, these new procedures frequently involve new forms of stress, side effects such as induction of genetic changes in the SE-derived and cryopreserved plant material. The examples of these new stress-inducing factors can be Picloram or NAA added instead of 2,4-D to proliferation medium or strong drying of ETs after application of the pregrowth-dehydration as a cryopreservation method (Hazubska-Przybył and Bojarczuk, 2008;Hazubska-Przybył et al, 2010). The long-term proliferation of ETs of both spruce species on the medium supplemented with Picloram during their maintenance under in vitro cultures was used in our present study.…”

“…In the case of P. omorika, only a few studies on the SE method have been carried out as of yet (Hazubska-Przybył and Bojarczuk, 2008;Mihaljević and Jelaska, 2005). Currently, it is known that both spruce species can be propagated using this technique, and that their embryogenic cultures can also be successfully stored in liquid nitrogen (LN; Hazubska-Przybył et al, 2010Find, 1998). These results will potentially allow for including Picea abies and P. omorika SE techniques into forest breeding programs and ornamental nurseries in the future.…”