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 fromimmature 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 di erent types of cytokinins (1 mg.L1) 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 e ect 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.L1), 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 di erent 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%

Cryopreservation of embryogenic tissues of hybrid firs: the effect of sorbitol on the tissue regrowth and postthaw recovery

vokMyynti MTT, Tietopalvelut 31600 Jokioine

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

© 2016 Institute of Botany, Slovak Academy of Sciences. 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.status: publishe

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

ConservePlants : an integrated approach to conservation of threatened plants for the 21st century

Even though plants represent an essential part of our lives offering exploitational, supporting and cultural services, we know very little about the biology of the rarest and most threatened plant species, and even less about their conservation status. Rapid changes in the environment and climate, today more pronounced than ever, affect their fitness and distribution causing rapid species declines, sometimes even before they had been discovered. Despite the high goals set by conservationists to protect native plants from further degradation and extinction, the initiatives for the conservation of threatened species in Europe are scattered and have not yielded the desired results. The main aim of this Action is to improve plant conservation in Europe through the establishment of a network of scientists and other stakeholders who deal with different aspects of plant conservation, from plant taxonomy, ecology, conservation genetics, conservation physiology and reproductive biology to protected area's managers, not forgetting social scientists, who are crucial when dealing with the general public.peer-reviewe