**2.1.6 Droplet method**

The droplet method was first reported by Schäfer-Menuhr et al. (1994, 1997) using potato apices. The operating procedure is the same for vitrification. However, the LS immersion protocol differs compared with that in the vitrification method. This cryopreservation method is shown in Fig. 5. After treatments by LS and PVS, plant samples are put on aluminum foil which is sterilized and cut small. One drop of PVS is dripped onto plant samples, and the whole aluminum foil is immersed in LN. The aluminum foil after cryopreservation is taken out from LN, and one drop of unloading solution supplemented with 1.0 mol/L sucrose is driiped onto to freezing samples. After rewarming, samples are

Fig. 4. The protocol of encapsulation-vitirication method (from Matsumoto et al., 1995)

The simplified encapsulation-vitrification method was first reported by Hirai & Sakai (2002) using shoot apices of sweet potato. The operating procedure in this method is the same as encapsulation-vitrification (see Fig. 4), however, the composition of LS differs. LS of simplified encapsulation-vitrification includes high-concentration glycerol (2.0 mol/L) and sucrose (1.6 mol/L), and the viscosity of LS is high. Although this method succeeded with sweet potato, there are some plant species which cannot be cryopreserved using high

The droplet method was first reported by Schäfer-Menuhr et al. (1994, 1997) using potato apices. The operating procedure is the same for vitrification. However, the LS immersion protocol differs compared with that in the vitrification method. This cryopreservation method is shown in Fig. 5. After treatments by LS and PVS, plant samples are put on aluminum foil which is sterilized and cut small. One drop of PVS is dripped onto plant samples, and the whole aluminum foil is immersed in LN. The aluminum foil after cryopreservation is taken out from LN, and one drop of unloading solution supplemented with 1.0 mol/L sucrose is driiped onto to freezing samples. After rewarming, samples are

**2.1.5 Simplified encapsulation-vitrification** 

concentration glycerol (Hirai & Sakai, 1999).

**2.1.6 Droplet method** 

moved from the cryotube, and recultured. In the droplet method, in order to make a plant sample cool quickly, Wesley-Smith et al. (2001) used not liquid nitrogen but a slush nitrogen (-210 oC) and an isopentane (-160 oC). In addition, the droplet method can reportedly obtain a high regrowth percentage after cryopreservation in tropical plants difficult to cryopreserve (Pennycooke & Towill, 2000, 2001; Leunufna & Keller, 2003; Panis et al., 2005).

Fig. 5. The protocol of Droplet method (from Schäfer-menuhr et al., 1997).
