**2.2.2 Treatment before cryopreservation**

Before cryopreservation, cold-acclimation and preculture are done, so survival percentages will increase after cryopreservation.

Cold-acclimation is a treatment by which plantlets are cultured at about 5 oC for one week to two months. However, Chang et al. (2000) reported that cold-acclimation was performed at - 1 oC in grass species (*Zoysia* and *Lolium* sp.). The freezing resistance of plant specimens reportedly increases by cold-acclimation (Chang et al., 2000). However, since coldacclimation cannot be adapted for a tropical plant, you should not perform this operation. Moreover, optimal acclimation periods differ by plant germplasms. In addition, prolonged cold-acclimation may curve and lower the survival percentage of plant specimens after cryopreservation. Therefore, I recommend that you closely consider the optimal cold acclimation period before trying cryopreservation.

Preculture is the treatment which gives plant cells or tissues dehydration tolerance. In many cases, plant samples are cultivated for 24~48 hours by culture medium supplemented with high-concentration the sucrose (0.3~0.7 mol/L). And some plant species are moved gradually from low to high concentration of sucrose medium (Niino et al., 1992; Niino & Sakai, 1992a,b; Suzuki et al., 1994; Niino et al., 1997). In addition, there are also cases in which glycerol (Matsumoto et al., 1998; Niino et al., 2003), DMSO (Fukai, 1990), or abscisic acid (ABA; Kendal et al., 1993; Tsukazaki et al.,2000) is mixed with a sucrose culture medium, and culture medium containing sorbitol without sucrose are used (Yamada et al., 1991; Maruyama et al., 2000). In many cases, room temperature is used for treatment (20~25 oC). However, some plant species can be processed by -1 oC (Chang et al., 2000) or 5 oC (Niino & Sakai, 1992a,b; Kuranuki & Sakai, 1995; Tanaka et al., 2004).
