Somatic polyembryogenesis in embryogenic cell masses of Piceaabies (Norway spruce) and Pinustaeda (loblolly pine) after thawing from liquid nitrogen

Abstract

We describe a method for the possible cryopreservation of embryogenic callus of Piceaabies and Pinustaeda at −196 °C and the regeneration of somatic embryos from thawed cells of subcultured embryonal–suspensor masses. Piceaabies and Pinustaeda were frozen without cryoprotective agent, in the presence of dimethyl sulfoxide (10%), or in a mixture of polyethylene glycol, glucose, and dimethylsulfoxide (10, 8, and 10% w/v, respectively). Cell masses placed in plastic vials or aluminum envelopes were frozen at 1 °C/min to −30 °C and then immersed for 10 min in liquid nitrogen. Cells were thawed rapidly and placed on modified MS subculture medium. Six to seven somatic embryos per gram of fresh weight were regenerated from each piece of frozen cell mass as compared with 12–13 embryos per gram from unfrozen cells. Post-thaw cell growth was inhibited initially by up to 5 weeks. Inhibition was reversed after the third 10-day subculture. Results suggest that the long-term storage of embryogenic cell lines in liquid nitrogen may be feasible for tree improvement programs in circumstances where testing of progeny may take several years.

Tissue Culture of Woody Plants and Its Relevance to Molecular Biology

“…Cryopreservation is a helpful tool for increasing the storage period of even synthetic seeds, and for international exchange of germplasm (Baj~, 1995b). Embryogenic conifer cultures (Kartha et al, 1988;Gupta et al, 1987;Klimazewska et al, 1992;Hargreaves and Smith, 1994;Sakai, 1995Find-Jens et al, 1998Park et al, 1998Park et al, ). (pence, 1996, seeds or isolated embryonic axes ofAzadirachta indica (Berjak and Dwnet, 1996), in vitro-grown shoot tips of cherry and sweet cherry (Niino et al, 1997) and shoot tips of silver birch (Ryyananen, 1998) have all been cryopreserved successfully.…”

Cryopreservation of Embryogenic Cultures of Conifers and Its Application to Clonal Forestry

“…Norway spruce ESM, after pretreatment with sucrose and dimethylsulphoxide (DMSO) and storage in liquid nitrogen for one hour, exhibited a lag-phase during re-establishment but no morphological abnormalities (Galerne and Dereuddre 1987). Gupta et al ( 1987) demonstrated embryo and germinant production from Norway spruce and loblolly pine embryogenic cultures after pretreatment with DMSO (D) or DMSO, PEG and glucose (D+P+G), followed in order by two D+P+G incubations then I 0 minutes immersion in liquid nitrogen. The cryopreservation protocol, which forms the basis of many current applications was developed by Kartha et a/.…”

Cryopreservation in liquid nitrogen of cultured navel organe (Citrus sinensis Osb.) nucellar cells and subsequent plant regeneration

“…In citrus also, plants regenerated from protoplasts, through somatic embryogenesis, showed phenotypic stability (Kobayashi, 1987). Because of this stability, embryogenic callus cultures have been widely used for cryopreservation (Ulrich et al 1982;Gupta et al 1987;Hahne & Lrrz 1987;Kartha et al 1988).…”