**2. Methods of cryopreservation of plant genetic resources**

In the section (2.1), I would like to introduce cryopreservation methods of plant genetic resources that have been developed. In the section (2.2), I would like to describe the approach when cryopreserving plant samples from the past literatures or my own experience in order to enhance the regrowth percentage after cryopreservation,

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are removed from a tube. After rewarming, samples are moved from the cryotube, and recultured. The cooling rate in this method is important. It differs from 0.5 oC/min to 50 oC/min with plant species and the size of the plant germplasm. However, in the case of the freezing speed of 2 oC/min or more, the regrowth after preservation tends to fall (Sugawara & Sakai, 1974; Uemura & Sakai, 1980). The disadvantage of this method is that there are many species for which the prefreezing method is not utilized at all. In addition, there are plant tissues which freeze to death partially, and cases in which the decrease in subsequent

This cryopreservation method was reported using samples of several species in the early 1990's. The advantage of this method is that researchers can cryopreserve without a special

The slow unprogrammed freezing is shown in Fig. 2. Plant tissues are added to the tube containing cryoprotectants. Tubes are treated for about 10 min at room temperature (25 oC), and are kept at -30 oC for 30~120 min. They are then immersed in LN thereafter. Cryopreserved tubes are warmed using hot water (40 oC) for 1~2 min, and cryoprotectants are removed from a tube. After rewarming, samples are moved from the cryotube, and recultured. In this cryopreservation method, mixtures of glycerol and sucrose or DMSO and sorbitol are used as cryoprotectants (Sakai et al., 1991; Niino et al., 1992; Maruyama et al., 2000). In this cryopreservation method, although 'naked' samples are used, Kobayashi et al. (2005) utilized cells encapsulated with alginate beads in the suspension cells of tobacco.

viability induced also exists (Grout & Henshaw, 1980; Haskins & Kartha, 1980).

**2.1.2 Slow unprogrammed freezing (also known as "simple freezing")** 

Fig. 2. The protocol of slow unprogrammed freezing (from Sakai et al., 1991).

programmable freezer, compared with slow programmed freezing.
