**2.1.4 Encapsulation-vitrification**

The encapsulation-vitrification method was reported first by Matsumoto et al. (1995) using shoot apices of *Wasabia japonica*, and then spread all over the world. The advantage of this method is that regrowth of plant germplasm after cryopreservation is markedly increased by encapsulating plant samples with alginate beads. The encapsulation of plant germplasms makes for less damage to samples during vitrification procedures (loading treatment and dehydration treatment). On the other hand, due to encapsulation-dehydration, treatment time becomes long compared with that of vitrification and the cryopreservation operation becomes complicated (for example, encapsulation).

The procedure for encapsulation-vitrification is shown in Fig. 4. Plant tissues are immersed in the calcium-free liquid medium supplemented with 0.4 mol/L sucrose, 30.0 g/L sodium alginate and glycerol (1.0~2.0 mol/L). The mixture (including a plant cell or tissue) was added drop by drop to the liquid medium containing 0.1 mol/L calcium chloride, forming beads about 5 mm in diameter. The above-mentioned liquid mediums (30.0 g/L sodium alginate and 0.1 mol/L calcium chloride) were adjusted by pH 5.7, but without plant growth regulators. Encapsulated specimens are added to the culture bottle containing LS for osmoprotection. Beads in the bottles are osmoprotected for 16 hours at room temperature (25 oC). LS is the liquid culture medium in which sucrose (0.75~0.8 mol/L) and the glycerol (2.0 mol/L) were contained. After loading, LS is removed from a bottle, and PVS is added newly for the dehydration of plant tissues. The same as with vitrification, the dehydration using PVS is performed at 0 oC in light of the toxicity to plant cells.

After dehydration of PVS, encapsulated samples are moved to a cryotube containing fresh PVS, and immersed in LN. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and the vitrification solution is removed from the tube. After removal of the solution, unloading solution (supplemented with 1.2 mol/L sucrose; pH 5.7) is added to a tube, and cryoprotectants are removed from plant tissues for 30 min at 25 oC. After unloading, samples are moved from the cryotube, and recultured.

Cryopreservation of Plant Genetic Resources 445

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

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

Dehydration was first reported by Uragami et al. (1990) using asparagus lateral buds. A dry technique is superior to vitrification in that it does not need to produce PVS. Therefore, there is no influence of medical toxicity at low cost. Problems of dehydration include ready influence of humidity on drying by air flow and dried samples are easily crushed with

The cryopreservation procedure is shown in Fig. 6. Plant tissues are put on the filter paper or nylon mesh sterilized and cut small. Samples are dehydrated by silica gel (Uragami et al., 1990) or air flow (Shimonishi et al., 1992; Kuranuki & Yoshida, 1996) before immersion in LN. It is reported that the optimal moisture of the sample is 10%~30% for survival after cryopreservation in the dehydration method (Uragami et al., 1990; Shimonishi et al., 1992; Kuranuki and Yoshida, 1996). After the dehydration, germplasms are moved to a cryotube and immersed in LN. Cryopreserved tubes are warmed at room temperature or using hot

et al., 2005).

**2.1.7 Dehydration** 

tweezers.

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