**2.1.7 Dehydration**

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 tweezers.

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

Cryopreservation of Plant Genetic Resources 447

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 &

A newly developed encapsulation-dehydration method was first reported by Sakai et al. (2000). The operating procedure is the same as for encapsulation-dehydration (see Fig. 7), however, the LS composition differs. LS of the newly developed encapsulation-dehydration includes a high concentration (2.0 mol/L) of glycerol besides sucrose. Therefore, the loading time of this method (1 hour) is shorter than that of encapsulation-dehydration (16 hours).

In this section, I introduce some approaches to increase regrowth of samples after

Before performing cryopreservation of plant samples, it is necessary to grasp the characteristics of the given plant species. For example, it is better to utilize encapsulation-

Sakai, 1995; Kami et al., 2005, 2007, 2008).

**2.2.1 Plant material** 

**2.1.9 Newly-developed encapsulation-dehydration** 

**2.2 Methods of improvement of cryopreservation efficiency** 

rewarming with past reports and actual experimental data I obtained.

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

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