Comparison of three techniques for cryopreservation and reestablishment of long-term Gentiana tibetica suspension culture.
Cryogenic storage of cell suspensions allows long-term maintenance of cultures. The main purpose of the study was to develop a successful cryogenic protocol for 10-year-old embryogenic cell suspensions of G. tibetica. We examined three techniques of freezing: (I) controlled-rate cooling with various cryoprotectants (0.1-0.5 M DMSO, 0.5-1.0 M sucrose, 0.5-1.0 M glycerol, 0.25-1.0 M proline) or preculture with 0.4 M sorbitol and cryoprotectants (0.065-0.1 M DMSO, 0.2-0.8 M proline), (II) vitrification (PVS2) and (III) encapsulation. Cell viability was assessed by the TTC test and biomass increase. After controlled-rate cooling the majority of cells were lethally damaged, with only 3% viability observed. Vitrification and encapsulation approaches were more effective, assuring high levels of post-thaw viability ca. 85% and 7%, respectively. The encapsulation procedure gave faster recovery of the culture suspension than did vitrification, and ensured culture homogeneity and embryogenic competence.
Mikuła
Anna
A
Botanical Garden, Center for Biological Diversity Conservation, Polish Academy of Sciences, Warsaw, Poland. amikula@ob.neostrada.pl
D018929:Cell Culture Techniques; D002455:Cell Division; D048430:Cell Shape; D048429:Cell Size; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods* / Q000592:standards*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004357:Drug Synergism; D005615:Freezing; D030012:Gentiana / Q000196:embryology*; D010084:Oxidation-Reduction; D012639:Seeds / Q000166:cytology* / Q000502:physiology; D013012:Sorbitol / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000378:metabolism; D013997:Time Factors
D003451:Cryoprotective Agents; D013778:Tetrazolium Salts; D013012:Sorbitol; C009591:triphenyltetrazolium