**2.1.9 Newly-developed encapsulation-dehydration**

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).

#### **2.2 Methods of improvement of cryopreservation efficiency**

In this section, I introduce some approaches to increase regrowth of samples after rewarming with past reports and actual experimental data I obtained.

## **2.2.1 Plant material**

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-

Cryopreservation of Plant Genetic Resources 449

Before cryopreservation, cold-acclimation and preculture are done, so survival percentages

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

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

I would like to explain this paragraph with actual experimental data I obtained. In vitrification, I examined the effect of exchange times of fresh PVS2 during a 60-min PVS2 loading treatment on shoot apices (*Cardamine yezoensis* Maxim.) immersed in LN using a vitrification protocol (Fig. 9). The shoot regeneration percentages after cryopreservation was enhanced up to 96.7% when two PVS2 exchanges were used. Moreover, above 80% of shoot regrowth was maintained also by three or more PVS2 exchanges. From this experiment, it became clear that the injury by too much dehydration and medical toxicity are not induced by the exchange of fresh vitrification solution. However, the increase in the exchange time of vitrification solution carries a complex risk of losing the shoot apex and operating. Therefore, I considered that even 2 exchanges during 60-min PVS2 loading treatment on

Since PVS2 at 0 oC has high viscosity and the circulation in the cryobial is poor, it is thought PVS2 around a shoot apex was diluted by the moisture flowing out of the plant tissue. Therefore, by exchanging for fresh PVS2, the dilution of PVS2 around a shoot apex was

Furthermore, adding an ice blocking agent to PVS reportedly enhances regeneration of

**2.2.2 Treatment before cryopreservation** 

acclimation period before trying cryopreservation.

**2.2.3 Treatment under cryopreservation** 

prevented and the dehydration maintained.

cryopreserved sample in recent years (Zhao et al., 2005).

(Niino & Sakai, 1992a,b; Kuranuki & Sakai, 1995; Tanaka et al., 2004).

shoot apices of *Cardamine yezoensis* was appropriate (Kami et al., 2010).

will increase after cryopreservation.

dehydration rather than vitrification for plant species which are subject to toxicity from cryoprotectants. Moreover, if you want to cryopreserve the plant germplasms readily susceptible to toxicity in DMSO with the vitrification method, it is better to use PVS3 rather than PVS2 as the vitrification solution.

Next, I would like to explain this paragraph with actual experimental data I obtained. In cryopreservation, the extracted size of plant material also becomes important. When plant tissues are greatly (3 mm x 3 mm) trimmed, the extraction labor will decline with small tissue size (1 mm x 1 mm). However, the regrowth percentage of large tissues after cryopreservation seems to decrease more than that of small tissues (Fig. 8; Kami et al., 2010). From previous peports, the reason is that the smaller the size of the extracted plant, the more the osmosis cryoprotectant decreases (Kim et al., 2004, 2005).

Fig. 8. Effects of excised apex size and exposure time to plant vitrification solution (PVS) on the regrowth of shoot apices immersed in liquid nitrogen (LN) using vitrification. Apices were dehydrated with two types of PVS at 0 oC for various lengths of time prior to cooling (Cryopreserved) or without cooling to -196 oC (Treated Control). The PVS in a cryovial was exchanged just after PVS loading treatment to prevent deterioration of PVS by a loading solution in this study. After cooling for 1hour in LN, rewarming apices were transplanted into regrowth medium. Values represent mean ± SE of three determinations. Differences in mean values of regrowth of treated control and cryopreseved apices with different letters are statistically significant (Tukey's HSD at *p*<0.05) in all data. (from Kami et al., 2010)

dehydration rather than vitrification for plant species which are subject to toxicity from cryoprotectants. Moreover, if you want to cryopreserve the plant germplasms readily susceptible to toxicity in DMSO with the vitrification method, it is better to use PVS3 rather

Next, I would like to explain this paragraph with actual experimental data I obtained. In cryopreservation, the extracted size of plant material also becomes important. When plant tissues are greatly (3 mm x 3 mm) trimmed, the extraction labor will decline with small tissue size (1 mm x 1 mm). However, the regrowth percentage of large tissues after cryopreservation seems to decrease more than that of small tissues (Fig. 8; Kami et al., 2010). From previous peports, the reason is that the smaller the size of the extracted plant, the

Fig. 8. Effects of excised apex size and exposure time to plant vitrification solution (PVS) on the regrowth of shoot apices immersed in liquid nitrogen (LN) using vitrification. Apices were dehydrated with two types of PVS at 0 oC for various lengths of time prior to cooling (Cryopreserved) or without cooling to -196 oC (Treated Control). The PVS in a cryovial was exchanged just after PVS loading treatment to prevent deterioration of PVS by a loading solution in this study. After cooling for 1hour in LN, rewarming apices were transplanted into regrowth medium. Values represent mean ± SE of three determinations. Differences in mean values of regrowth of treated control and cryopreseved apices with different letters are statistically significant (Tukey's HSD at *p*<0.05) in all data. (from Kami et al., 2010)

more the osmosis cryoprotectant decreases (Kim et al., 2004, 2005).

than PVS2 as the vitrification solution.
