Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Med icago sativa L.) cells and study the physiological changes occurring in cel ls during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (f all-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiat ed regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-t reatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid NZ ( EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of t he cv. 5262 cells after cryopreservation was two- to three-fold greater tha n that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2 d egrees C) improved viability after cryopreservation. The addition of 7.6 mu M ABA to the B5 medium enhanced viability in ED but did not improve cell cr yopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold -acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased conc entration of cell protein and an altered protein composition. Suspension cu ltures were re-initiated from both ED and EDN treatments by transferring be ads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures , whereas EDN cells needed four or five subcultures to resume rapid growth.