**2.1.3 Vitrification**

The vitrification method has been the major cryopreservation method since Uragami et al. (1989) developed it using asparagus culture cells. This cryopreservation method is shown in Fig. 3. Plant tissues are added to the tube containing the loading solution (LS) for the osmoprotection. Beads in tubes are osmoprotected for about 30 min at room temperature (about 25 oC). LS is the liquid culture medium in which sucrose (0.4 mol/L) and the glycerol (2.0 mol/L) were contained. After loading, LS is removed from a tube, and new vitrification solution is added for the dehydration of plant tissues.

Fig. 3. The protocol of vitirication method (from Sakai et al., 1990).

Many cryoprotectants are dissolved in the vitrification solution, and the optimal dehydration time using the solution changes greatly with treatment temperature.

In many cases, the dehydration using the vitrification solution is performed at 0 oC by the reason of the toxicity to plant cells. Plant Vitrification Solution 2 (PVS2; Sakai et al., 1990) is utilized most as the vitrification solution. Besides PVS2, there are many vitrification solutions. Please refer to Table 1 for the composition. They are immersed in LN after that. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and the vitrification solution is removed from a tube. After the removal of vitrification solution, unloading solution (the liquid medium supplemented with 1.2 mol/L sucrose) is added to a tube, and cryoprotectants are removed from plant tissues for 30 min at 25 oC. In many cases, the above-mentioned liquid mediums (LS, PVS and unloading solution) were adjusted by pH

The vitrification method has been the major cryopreservation method since Uragami et al. (1989) developed it using asparagus culture cells. This cryopreservation method is shown in Fig. 3. Plant tissues are added to the tube containing the loading solution (LS) for the osmoprotection. Beads in tubes are osmoprotected for about 30 min at room temperature (about 25 oC). LS is the liquid culture medium in which sucrose (0.4 mol/L) and the glycerol (2.0 mol/L) were contained. After loading, LS is removed from a tube, and new vitrification

**2.1.3 Vitrification** 

solution is added for the dehydration of plant tissues.

Fig. 3. The protocol of vitirication method (from Sakai et al., 1990).

Many cryoprotectants are dissolved in the vitrification solution, and the optimal

In many cases, the dehydration using the vitrification solution is performed at 0 oC by the reason of the toxicity to plant cells. Plant Vitrification Solution 2 (PVS2; Sakai et al., 1990) is utilized most as the vitrification solution. Besides PVS2, there are many vitrification solutions. Please refer to Table 1 for the composition. They are immersed in LN after that. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and the vitrification solution is removed from a tube. After the removal of vitrification solution, unloading solution (the liquid medium supplemented with 1.2 mol/L sucrose) is added to a tube, and cryoprotectants are removed from plant tissues for 30 min at 25 oC. In many cases, the above-mentioned liquid mediums (LS, PVS and unloading solution) were adjusted by pH

dehydration time using the solution changes greatly with treatment temperature.


5.7~5.8, but without plant growth regulators. After unloading, samples are removed from the cryotube, and recultured.

Table 1. Components of major plant vitrification solutions. Components of three plant vitrification solutions are referred from previous reports (Uragami et al., 1989; Sakai et al., 1990; Nishizawa et al., 1993).
