Enhanced Cell Viability for the Efficient Cryopreservation of Transgenic Rice Cells

초록

Transgenic rice cell suspension cultures (Oryza sativa L.) have been utilized to produce recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). General long-term preservation method for plant cells has been known to be the repeated subcultures. One drawback of this method is the genetic instability of transformed cell lines. For that reason, a cryopreservation method was developed for the transgenic rice cells. Dimethyl sulfoxide (DMSO) has been known to be an effective cryoprotectant in freezing steps, but the cryopreserved cells are damaged during thawing procedures. To solve this problem, an optimal method was designed by using various cryoprotectant mixtures in freezing step and the cryoprotectants were removed by negative pressure in thawing steps. Optimal concentration of acryoprotectants was found to be 1 M sucrose, 0.5 M glycerol and 0.5 M DMSO, respectively. The cell viability was 57.3% in the optimized combination after 7 days of cryopreservation. To remove toxic cryoprotectants in thawing steps, the time required for negative pressure treatment was found to be 2 min and cell viability was 84.7%. In addition, the cells were recovered to callus efficiently in the solid medium. After 3 weeks, regeneration of cells was confirmed by using cell morphology and increase of cell mass. In conclusion, we could enhance the cell viability after the cryopreservation of transgenic rice cells by optimizing cryoprotectants-related steps.