**2.2.4 Treatment after cryopreservation**

In cryopreservation of plant genetic resources, regeneration after rewarming is the key. Surviving cells or tissues after cryopreservation readily succumb due to different environmental agents because they have been injured by the dehydration or temperature change during the cryopreservation procedure. Moreover, when plant specimens were injured by the cryopreservation process, polyphenol can be produced. Thus, this may threaten the survival of plant specimens after cryopreservation. In that case, regeneration of tissues after preservation reportedly increased when activated charcoal (Bagniol & Engelmann, 1992) and polyvinyl pyrrolidone (Niino et al., 2003), an adsorbent of polyphenol, was mixed with a culture medium.

In recent years, it is also reported that regrowth percentages of rewarming tissues increased by mixing surfactant with regrowth medium (Anthony et al., 1996; Niu et al., 2010). Therefore, special consideration must be given to certain plant species. From previous reports, the regrowth after preservation increases sharply also by decreasing NH4+ concentration in a culture medium (Niino et al., 1992a, 1992b; Suzuki et al., 1994; Pennycooke & Towill, 2001).

Next, I would like to explain this paragraph with actual experimental data I obtained. I examined the effects of various nutrient media (Table 2) on regrowth of cryopreserved

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

In cryopreservation of plant genetic resources, regeneration after rewarming is the key. Surviving cells or tissues after cryopreservation readily succumb due to different environmental agents because they have been injured by the dehydration or temperature change during the cryopreservation procedure. Moreover, when plant specimens were injured by the cryopreservation process, polyphenol can be produced. Thus, this may threaten the survival of plant specimens after cryopreservation. In that case, regeneration of tissues after preservation reportedly increased when activated charcoal (Bagniol & Engelmann, 1992) and polyvinyl pyrrolidone (Niino et al., 2003), an adsorbent of

In recent years, it is also reported that regrowth percentages of rewarming tissues increased by mixing surfactant with regrowth medium (Anthony et al., 1996; Niu et al., 2010). Therefore, special consideration must be given to certain plant species. From previous reports, the regrowth after preservation increases sharply also by decreasing NH4+ concentration in a culture medium (Niino et al., 1992a, 1992b; Suzuki et al., 1994;

Next, I would like to explain this paragraph with actual experimental data I obtained. I examined the effects of various nutrient media (Table 2) on regrowth of cryopreserved

(Tukey's HSD at *p*<0.05) in each treatments. (from Kami et al., 2010)

**2.2.4 Treatment after cryopreservation** 

polyphenol, was mixed with a culture medium.

Pennycooke & Towill, 2001).

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; Kami et al., 2010).


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

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. (from Kami et al., 2010)

Cryopreservation of Plant Genetic Resources 453

Kami, D.; Uenohata, M., Suzuki, T. & Oosawa, K. (2008). Cryopreservation of black

Kami, D.; Shi, L., Sato, T., Suzuki, T. & Oosawa, K. (2009). Cryopreservation of shoot apices

Kami, D.; Kido, S., Otokita, K., Suzuki, T., Sugiyama, K. & Suzuki, M. (2010).

Kartha,K.K. (1985). *Cryopreservation of Plant Cells and Organs,* CRC Press, ISBN 0-8493-6102-8,

Kendall, E.J.; Kartha, K.K., Qureshi, J.A. & Chermak, P. (1993). Cryopreservation of

Kim, H.H.; Kim, J.B., Baek, H.J., Cho, E.G., Chae, Y.A. & Engelmann, F. (2004). Evolution of

Kim, J.B.; Kim, H.H., Baek, H.J., Cho, E.G., Kim, Y.H. & Engelmann, F. (2005). Changes in

Kumu, Y.; Harada, T. & Yakuwa, T. (1983). Development of a whole plant from a shoot tip

*Hokkaido University*, Vol.61, No.3, (May 1983), pp. 285-294, ISSN 0018-344X Kuranuki, Y. & Sakai, A. (1995). Cryopreservation of *in vitro*-grown shoot tips of tea

Kuranuki, Y. & Yoshida, S. (1996). Differential responses of embryogenic axes and

Leunufna, S. & Keller, E.R.J. (2003). Investigating a new cryopreservation protocol for yams

Maruyama, E., Tanaka, T., Hosoi, Y., Ishii, K. & Morohoshi, N. (2000). Embryogenic cell

Matsumoto, T.; Sakai, A., Takahashi, C. & Yamada, K. (1995). Cryopreservation of *in vitro*-

Vol.120, No.2, (August 2008), pp. 84-88, ISSN0304-4238

pp.119-126, ISSN 1340-7902 (in Japanese with English summary)

*Cell Reports*, Vol.12, No.2, (Octobar 1992), pp. 89-94, ISSN 0721-7714

*Letters*, Vol.25, No.1, (October 2003), pp. 91-100, ISSN 0143-2044

*Biotechnology*, Vol.22, No.2, (March 2005), 105-112, ISSN 1342-4580

46, No.2, pp. 149-154, (March 1996), ISSN 1347-3735

Vol.17, No.4, (April 2000), 281-296, ISSN 1342-4580

2008), ISSN 0143-2044

Florida, U.S.A.

352, ISSN 0143-2044

0721-7714

chokeberry *in vitro* shoot apices. *Cryo-Letters*, Vol.29, No.3, pp.209-216, (January

of hawthorn *in vitro* cultures originating from East Asia. *Scientia Horticulturae*,

Cryopreservation of shoot apices of *Cardamine yezoensis in vitro*-cultures by vitrification Method. *Cryobiology and Cryotechnology*, Vol.56, No.2, (September 2010),

immature spring wheat zygotic embryos using an abscisic acid pretreatment. . *Plant* 

DMSO concentration in garlic shoot tips during a vitrification procedure, *Cryo-*

sucrose and glycerol content in garlic shoot tips during freezing using PVS3 solution, *Cryo-Letters*, Vol.26, No.2, (December 2004), pp. 103-112, ISSN 0143-2044 Kobayashi, T.; Niino, T. & Kobayashi, M. (2005). Simple cryopreservation protocol with an

encapsulation technique for tabacco BY-2 supension cell cultures. *Plant* 

of *Asparagus officinalis* L. frozen to –196oC. *Journal of the Faculty of Agriculture,* 

(*Camellia sinensis*) by vitrification. *Cryo-Letters*, Vol.16, No.4, (April 1995), pp. 345-

cotyledons from tea seeds to desiccation and cryoexposure. *Breeding Science,* Vol.

(*Discorea* spp.). *Plant Cell Reports*, Vol.21, No.12, (April 2003), pp. 1159-1166, ISSN

culture, protoplast regeneration, cryopreservation, biolistic gene transfer and plant renegeration in Japanese cedar (*Cryptomeria japonica* D. Don). *Plant Biotechnology*,

grown apical meristems of wasabi (*Wasabia japonica*) by encapsulation-vitrification method. *Cryo-Letters*, Vol.16, No.2, (November 1994), pp. 189-196, ISSN 0143-2044
