**2.1.8 Encapsulation-dehydration**

The encapsulation-dehydration method was first reported by Fabre & Dereuddre (1990) using shoot apices of potato, and spread worldwide the same way as vitrification and encapsulation-vitrification. This method excels that of dehydration in that regrowth of plant germplasm after cryopreservation is markedly increased by encapsulating plant samples with alginate beads. In addition, encapsulated samples are difficult to be crushed with tweezers compared with the dehydration method.

The encapsulation-dehydration procedure is shown in Fig. 7. Plant tissues are immersed in a calcium-free liquid medium supplemented with 0.4 mol/L sucrose and 30.0 g/L sodium alginate. 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 5.7~5.8, but without plant growth regulators. Encapsulated germplasms are added to the culture bottle containing LS for the osmoprotection. Beads in the bottles are osmoprotected for 16 hrs at room temperature (25 oC). LS is the liquid culture medium in which sucrose (0.75~0.8 mol/L) is contained. After loading, LS is removed from the bottle. Loaded samples are put on sterilized filter papers, and samples are dehydrated by silica gel for 3~7 hours before immersion in LN. After dehydration by silica gel, encapsulated samples are moved to a cryotube, and immersed in LN. Cryopreserved tubes are warmed using hot water (40 oC) for 1 ~ 2 min. After rewarming, samples are moved from the cryotube, and recultured.

water (40 oC) for 1 ~ 2 min. After rewarming, samples are removed from the cryotube, and

Fig. 6. The protocol of Dehydration method (from Uragami et al., 1990).

The encapsulation-dehydration method was first reported by Fabre & Dereuddre (1990) using shoot apices of potato, and spread worldwide the same way as vitrification and encapsulation-vitrification. This method excels that of dehydration in that regrowth of plant germplasm after cryopreservation is markedly increased by encapsulating plant samples with alginate beads. In addition, encapsulated samples are difficult to be crushed with

The encapsulation-dehydration procedure is shown in Fig. 7. Plant tissues are immersed in a calcium-free liquid medium supplemented with 0.4 mol/L sucrose and 30.0 g/L sodium alginate. 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 5.7~5.8, but without plant growth regulators. Encapsulated germplasms are added to the culture bottle containing LS for the osmoprotection. Beads in the bottles are osmoprotected for 16 hrs at room temperature (25 oC). LS is the liquid culture medium in which sucrose (0.75~0.8 mol/L) is contained. After loading, LS is removed from the bottle. Loaded samples are put on sterilized filter papers, and samples are dehydrated by silica gel for 3~7 hours before immersion in LN. After dehydration by silica gel, encapsulated samples are moved to a cryotube, and immersed in LN. Cryopreserved tubes are warmed using hot water (40 oC) for 1 ~ 2 min. After rewarming, samples are moved from the cryotube, and recultured.

**2.1.8 Encapsulation-dehydration** 

tweezers compared with the dehydration method.

recultured.

Fig. 7. The protocol of Encapsulation-dehydration method (from Fabre & Dereuddre, 1990).

In encapsulation-dehydration, the addition of glycerol besides sucrose in LS reportedly enhances the regrowth percentage of cryopreserved samples. The optimal concentration of glycerol in LS is 0.5~2.0 mol/L for regrowth of cryopreserved specimens (Matsumoto & Sakai, 1995; Kami et al., 2005, 2007, 2008).
