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Embryogenic tissues of hybrid firs ( Abies alba  ×  A. cephalonica ,  Abies alba  ×  A. numidica ) have been cryopreserved using a slow-freezing method. The cryotolerance of six cell lines initiated from immature or mature zygotic embryos was tested. Following sorbitol (0.5 M) and DMSO (5%) pretreatments the samples were slowly frozen at a rate of 1°C/min, plunged into liquid nitrogen and stored for 1 year. Post-thaw regeneration ocurred in all the six tested cell lines with recovery frequencies ranging from 100% (cell lines AC1, AC2, AC78, AN72), 90% (cell line AC2) to 44.4% (cell line AC79). Fresh and dry mass accumulation of cryopreserved tissues evaluated three month after thawing was identical to that of control (non-cryopreserved tissues without pretreatment). The cryopreservation procedure resulted in disintegration of bipolar structure of somatic embryos. The long vacuolised suspensor cells almost completely disrupted and the meristematic embryonal cells survived cryopreservation. In the post-thaw period, repeated cell divisions of meristematic cells led to formation of new cell clusters and their vacuolisation resulted in polarisation and finally to the formation of bipolar structures and somatic embryos.

Tissue regeneration of Abies embryogenic cell lines after 1 year storage in liquid nitrogen