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

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Abstract
We describe a method for the possible cryopreservation of embryogenic callus of Picea abies and Pinus taeda at -196.degree. C and the regeneration of somatic embryos from thawed cells of subcultured embryonal-suspensor masses. Picea abies and pinus taeda 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.degree. C/min to -30.degree. 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 cells 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 circumustances where testing of progeny may take several years.