**8. Comparison of different cryopreservation methods**

The bases for achieving high regeneration after plant cryopreservation using a vitrification method needs to observe the time fulfilment in dehydrating procedures and prevention of

Comparison of Cryopreservation Methods of

genetic stability after usage of this method.

**9. Conclusion** 

Vegetatively Propagated Crops Based on Thermal Analysis 353

Several procedures of cryopreservation of fruit trees germplasm, especially apple tree were evaluated, including two-step cryopreservation with controlled ice nucleation (Niino *et al*., 1992; Niino & Sakai, 1992; Seufferheld *et al*., 1999; Zhao *et al*., 1999), and the type of vitrification method of extirpated *in vitro* cultures. The procedures were adjusted and corrected according to the thermal methods for determination of ice nucleation (Tyler *et al*., 1988), glass transitions in plant material, and exothermic and endothermic characteristics of plant buds. A combination of encapsulation-dehydration cryoprotocol was chosen as the most appropriate system of *in vitro* cultures (Chang & Reed, 2001). The reaction of selected cultivars was different on *in vitro* sub-cultivation and subsequent cryopreservation protocol. Many plants from the temperate and tropical region were successfully cryopreserved by the encapsulation-dehydration method, which belongs to cryopreservation methods. The ecapsulation-dehydration cryopreservation procedure is based on encapsulating shoot tips of pretreated *in vitro* plants with subsequent dehydration either in sterile air flow (Benson *et al*., 1996; Dereuddre *et al*., 1991) or above silicagel (Grospietsch *et al*., 1999). Dehydrated beads with encapsulated shoot tips are in most cases plunged directly into liquid nitrogen or slowly frozen in programmable freezers (Fabre & Dereuddre, 1990); Zhao *et al*., 2001). Rewarming proceeds either slowly by placing beads on Petri dishes or the cryotubes are placed in a water bath of temperature ranging from 25 to 45 °C for several minutes (Gupta & Reed, 2006; Matsumoto & Sakai, 1995). Survival and regeneration of shoot tips are evaluated after placing the re-warmed beads on cultivation medium which can be compound modified by phytohormones (their combination and concentration), to stimulate proliferation or eliminate the phenolic compounds (Paulet *et al*., 1993). The medium can be softer in some cases to allow an easier regeneration from beads (Reed *et al*., 2006) or even the explants can be extracted from the beads (Niino & Sakai, 1992). The importance of direct explant regeneration to the new plants without callus inter phase is important for the

The four main crops (garlic, potato, hop and apple tree) have been cryopreserved in the Czech Plant Cryobank. The methods of cryopreservation are based on cryoprotocol of the vitrification procedures (encapsulation-dehydration, dehydration by vitrification solution and a modified ultra–rapid freezing method based on preconditioning of the plant shoot tips on an osmotic solution). Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases (Engelmann, 2011). We have started the routine cryopreservation with Czech potato genotypes in collaboration with Potato Research Institute Ltd., Havlíčkův Brod. The other three crops are supported by specialized companies (*Allium* genera by the Gene Bank Olomouc branch, the Research and Breeding Institute of Pomology at Holovousy and the Hop Research Institute). The Czech Plant Cryobank operates as a safe duplicate to repositories of germplasm kept in field or *in vitro* conditions. In this way the Czech Plant Cryobank in Prague joined the effort of the potato cryobank in Germany (Keller *et al*., 2008), and Korea (Kim *et al*., 2006). Currently three of the EU countries (The Czech Republic, Germany and Poland) involved in the maintenance of national vegetatively propagated *Allium* collections are developing the methodology for cryopreservation (project

tissue damage by chemical toxicity of the cryoprotectants. Otherwise, plant parts in solutes can be injured by the cryoprotectant, by strong osmotic stress during the cryoprotective solution treatment. The time for a high regeneration rate of plants after cryopreservation must be optimized. During this time the explants are dipped in the vitrification solutions, at the optimal temperature, which is involved in the procedure (Condello *et al*., 2011; Faltus & Zamecnik, 2009; Sakai & Engelmann, 2007; Zámečník *et al*., 2007).

The temperature-induced glasses-the point at which this occurs, is called the glass transition temperature (Tg) - molecular motion nearly ceases and the liquid becomes a glassy solid. Vitrification of cells and tissues is a physical process which avoids intracellular ice crystallization during ultra-rapid freezing by the transition of the aqueous solution of the symplast into an amorphous glassy state of the cells. As a consequence of the vitrification process, plant tissues are protected from the damage and remain viable during their longterm storage at –196 °C.

Experimental determination of glassy state in plants is complicated by the endothermic reaction overlapping with the glass transition. Ice crystallization as the first-order reaction has discontinuous change in heat capacity contrary to glass transition, which is characterized by heat capacity change. The main problem is to distinguish the endothermic reaction and the glass transition temperature during the measurement of the thermal events. Thermal analysis methods of glass transition temperature and temperature of ice crystals melting in plant tissues were determined. Standard Differential Scanning Calorimetry (DSC) method and Temperature Modulated Differential Scanning Calorimetry (TMDSC) were usually used (Condello *et al*., 2011; Zámečník & Faltus, 2009).


Table 5. Cryopreservation methods used in this study. Results obtained by cryopreservation methods with a high regeneration rate were presented only: \*\* - high regeneration rate; \* tested; NT- not tested; (§)-PVS2 was used.

The vitrification method, based on involvement of biological glass, requires a highly concentrated solution of cryoprotectant (from 5 to 8 M), at which the cells are osmotically dehydrated to a certain level. This level of dehydration is characterized by no frost-heaving of water and with a minimum, or no production of ice crystals. The cells treated by cryoprotectant are then vitrified before or during immersion into liquid nitrogen (Sakai *et al*., 1991). Vitrification belongs to new well-developed procedures supplying frost dehydration of cells pursued at a low temperature. Namely, it removes most of the freezable water through the exposure of the plant shoot tips to a highly concentrated vitrification solution at temperatures above the freezing point.

Several procedures of cryopreservation of fruit trees germplasm, especially apple tree were evaluated, including two-step cryopreservation with controlled ice nucleation (Niino *et al*., 1992; Niino & Sakai, 1992; Seufferheld *et al*., 1999; Zhao *et al*., 1999), and the type of vitrification method of extirpated *in vitro* cultures. The procedures were adjusted and corrected according to the thermal methods for determination of ice nucleation (Tyler *et al*., 1988), glass transitions in plant material, and exothermic and endothermic characteristics of plant buds. A combination of encapsulation-dehydration cryoprotocol was chosen as the most appropriate system of *in vitro* cultures (Chang & Reed, 2001). The reaction of selected cultivars was different on *in vitro* sub-cultivation and subsequent cryopreservation protocol.

Many plants from the temperate and tropical region were successfully cryopreserved by the encapsulation-dehydration method, which belongs to cryopreservation methods. The ecapsulation-dehydration cryopreservation procedure is based on encapsulating shoot tips of pretreated *in vitro* plants with subsequent dehydration either in sterile air flow (Benson *et al*., 1996; Dereuddre *et al*., 1991) or above silicagel (Grospietsch *et al*., 1999). Dehydrated beads with encapsulated shoot tips are in most cases plunged directly into liquid nitrogen or slowly frozen in programmable freezers (Fabre & Dereuddre, 1990); Zhao *et al*., 2001). Rewarming proceeds either slowly by placing beads on Petri dishes or the cryotubes are placed in a water bath of temperature ranging from 25 to 45 °C for several minutes (Gupta & Reed, 2006; Matsumoto & Sakai, 1995). Survival and regeneration of shoot tips are evaluated after placing the re-warmed beads on cultivation medium which can be compound modified by phytohormones (their combination and concentration), to stimulate proliferation or eliminate the phenolic compounds (Paulet *et al*., 1993). The medium can be softer in some cases to allow an easier regeneration from beads (Reed *et al*., 2006) or even the explants can be extracted from the beads (Niino & Sakai, 1992). The importance of direct explant regeneration to the new plants without callus inter phase is important for the genetic stability after usage of this method.
