**2.2.3 Treatment under cryopreservation**

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

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 prevented and the dehydration maintained.

Furthermore, adding an ice blocking agent to PVS reportedly enhances regeneration of cryopreserved sample in recent years (Zhao et al., 2005).

Cryopreservation of Plant Genetic Resources 451

apices (*Cardamine yezoensis* Maxim.). It was demonstrated that 4-fold dilution of inorganic salts of Murashige and Skoog's medium (1/4MS) or Woody Plant medium (WPM) as basal medium resulted in higher regrowth percentages (both 66.7%) than six other media (Fig. 10;

NO3- 45.39 22.70 11.35 5.67 0.79 26.77 30.25 6.30 NH4+ 20.61 10.31 5.15 2.58 0.00 2.33 7.01 5.00

Table 2. Compositions of eight types of nutrient medium for the regrowth of cryopreserved

Fig. 10. Effects of nutrient media on the regrowth of shoot apices immersed in LN using vitrification. Apices were dehydrated with PVS2 at 0oC for 60-mins prior to immersion in LN. The PVS2 in a cryovial was exchanged once just after PVS2 loading treatment. After cooling for 1hour in LN, rewarming apices were transplanted into 8 types of basal medium. Values represent mean ± SE of three determinations. Differences in mean values of regrowth with different letters are statistically significant (Tukey's HSD at *p*<0.05) in each treatments.

3- 1.25 0.62 0.31 0.16 0.12 1.09 2.94 1.25 K+ 20.05 10.02 5.01 2.51 1.63 24.73 30.94 12.61 Ca2+ 2.99 1.50 0.75 0.37 1.27 1.02 1.13 3.01 Mg2+ 3.07 3.07 0.77 0.28 5.98 2.08 1.54 3.07

1 1/2 1/4 1/8

Murashige & Skoog (MS) White B5 N6 WPM

Kami et al., 2010).

Component (mmol/L)

PO4

shoot apices

(from Kami et al., 2010)

Fig. 9. Effects of exchange times of PVS2 during 60-min PVS2 loading treatment on shoot apices immersed in LN using vitrification. 2 ml of fresh PVS2 were exchanged at 0oC for 60 mins prior to cooling. A PVS2 exchange just after PVS2 loading treatment was not counted as the exchanging time of PVS2 in this study. After cooling for 1hour in LN, rewarming apices were transplanted into 1/4MS. Values represent mean ± SE of three determinations. Differences in mean values of regrowth with different letters are statistically significant (Tukey's HSD at *p*<0.05) in each treatments. (from Kami et al., 2010)
