Physiological, Cytological and Biochemical Stability of Medicago Sativa L. Cell Culture After 27 Years of Cryogenic Storage

BACKGROUND: The efficiency of long-term cryogenic storage to prevent somaclonal variations in plant cell cultures and retain their major cytogenetic and biochemical traits remains under debate. In particular, it is not clear how stress conditions associated with cryopreservation, such as low temperature, dehydration and toxic action of some cryoprotectants (DMSO in particular), affect post-storage regrowth and genetic integrity of cell samples. OBJECTIVE: We assessed growth, cytogenetic and biochemical characteristics of the peroxidase-producing strain of Medicago sativa L. cell culture recovered after 27 years of cryogenic storage as compared to the same culture before cryopreservation. MATERIALS AND METHODS: In 1984, M. sativa L. cell culture was cryopreserved using programmed freezing and 7% DMSO as a cryoprotectant. In 2011, after rewarming in a water bath at 40°C for 90 s, cell culture was recovered and proliferated. Viability, growth profile, mitotic index, ploidy level, peroxidase activity and cell response to hypothermia and osmotic stress were compared between the recovered and the initial cell cultures using the records available from 1984. RESULTS: Viability of alfalfa cell culture after rewarming was below 20% but it increased to 80% by the 27^th subculture cycle. Recovered culture showed higher mitotic activity and increased number of haploid and diploid cells compared to the initial cell line. Both peroxidase activity and response to abiotic stress in the recovered cell culture were similar to that of the initial culture. CONCLUSION: Cryopreservation by programmed freezing was effective at retaining the main characteristics of M. sativa undifferentiated cell culture after 27 years of storage. According to available data, this is longest period of successful cryopreservation of plant cell cultures reported so far. After storage, there was no evidence that DMSO had any detrimental effect on cell viability, growth or cytogenetics.