Recovery of cytogenetic and physiological characteristics of a population of alfalfa cells after cryogenic storage

摘要

After 27 years of storage in liquid nitrogen, we managed to restore the growth of suspension cell culture of alfalfa (Medicago sativa L.). The influence of factors affecting viability of alfalfa cells in vitro was investigated together with cytogenetic and physiological characteristics after their storage at the temperature of liquid nitrogen (-196A degrees C). As a result of freezing/thawing, approximately 80% of predominantly polyploid cells died, and osmotic stress was the main injurious agent. Thus, after cryogenic storage, cell culture mainly consisted of hypo and hyper haploid and hypo and hyper diploid cells. After 35 growth cycles, the restored population reached a dynamic equilibrium between the cells of different levels of ploidy with modal class (accounting for more than 50%) of polyploid cells. The strain under investigation is notable for a high activity of peroxidase (PO). After cryogenic storage, PO activity considerably decreased; however, during 35 growth cycles, enzyme activity gradually rose to the level characteristic of original cell culture prior to freezing. In order to protect the cells from endogenous ice nucleation, we used cryoprotector dimethyl sulfoxide (DMSO). It was found that DMSO was not efficient at a concentration of 7% for cell protection against osmotic stress and exerted a moderate antiradical influence in respect to superoxide anion but improved viability of recovered cells and affected activity of PO and aldehyde reductase and the level of lipid peroxidation.