Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed

Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed

Physiological and Structural Aspects of In Vitro Somatic Embryogenesis in Abies alba Mill

Initiation of somatic embryogenesis from immature zygotic embryos, long-term maintenance of embryogenic tissue in vitro or by cryopreservation, as well as maturation, of somatic embryos of Abies alba Mill. are reported in this study. For the initiation of embryogenic tissues, a DCR medium containing different types of cytokinins (1 mg.L−1) were tested. During three consecutive years, 61 cell lines were initiated out of 1308 explants, with initiation frequencies ranging between 0.83 and 13.33%. The type of cytokinin had no profound effect on the initiation frequency within one given year. Microscopic observations revealed presence of bipolar somatic embryos in all initiated embryogenic tissues. Besides the typical bipolar somatic embryos, huge polyembryonal complexes, as well as “twin” embryos, were observed. Maturation of somatic embryos occurred on a DCR medium supplemented by abscisic acid (10 mg.L−1), polyethylene glycol (PEG-4000, 7.5%) and 3% maltose. The maturation capacity was cell-line dependent. All of the four tested cell lines produced cotyledonary somatic embryos, though at different quantities, of 16 to 252 per g of fresh weight. After germination, seedlings developed, but their further growth soon stopped after the formation of a resting bud. Altogether, seven cell lines were cryopreserved, using the slow-freezing technique. After rewarming, all tested cell lines showed regrowth rates between 81.8 and 100%status: publishe