**3.1 Cryoprotectants involved in vitrification method**

The cryopreservation method using a vitrification solution was first described by (Luyet, 1937). The vitrification solutions were firstly named, according to the first author of the publication and later the vitrification solutions have abbreviated names from Plant Vitrification Solution (PVS) with a number according to the time of their first appearance in the literature. The main ones are Luyet (1937), Fahy (1985), Steponkus (Langis & Steponkus, 1990), PVS1 (Uragami *et al*., 1989; Towill, 1990), PVS2 (Suzuki *et al*., 2008), PVS3, PVS4, PVS5 (Nishizawa *et al*., 1993), VS6 (Liu *et al*., 2004a), PVSL (Liu *et al*., 2004b) VSL (Suzuki *et al*., 2008), with different concentration and combination of the main four components: dimethylsulfoxide, sucrose, glycerol and ethylene glycol. The increased efficiency of vitrification methods was achieved by treating plants in the pre-cultivation step before cryopreservation of plant shoot tips in so called Loading Solution (LS) (Dumet *et al*., 2002; Matsumoto & Sakai, 1995; Sakai *et al*., 1991; Sakai & Engelmann, 2007). The cryoprotective substances should fulfil several basic parameters, such as cell permeability, viscosity, toxicity and the minimum concentration necessary for the vitrification, which eliminates the formation of ice crystals.

Cryoprotective substances help to ensure the stability of membranes and enzymes in the subsequent dehydration by vitrification solutions and to avoid the formation of ice crystals (Kartha & Leung, 1979; Kim *et al*., 2006). The samples are exposed to a several hour-long treatment by some cryoprotective substances, and then they are plunge-frozen in liquid nitrogen. The effect of cryoprotective solution composition for plant regeneration was studied in different plant species (Ellis *et al*., 2006; Kim *et al*., 2004; Kim *et al*., 2009; Tanaka *et al*., 2004).

In the most recent approaches to the garlic cryopreservation, vitrification method can be induced by treating the shoot tips of plantlets with a highly concentrated a mixture of glycerol and sucrose. (Nishizawa *et al*., 1993) developed Plant Vitrification Solution 3 (PVS3) with 50% glycerol (w/v) and 50% sucrose (w/v) in water. It is noteworthy that, following these procedures, the plant specimens can be directly plunged into liquid nitrogen, where they can be stored for an indefinite period of time without undergoing the risks of contamination or genetic alterations.

#### **3.2 Methods based on dehydration**

Potato (*Solanum tuberosum* L.) is a plant species sensitive to frost temperatures. Cryoprotocol for potato has to solve the problem of how to overcome temperature between 0 °C and –130 °C during cooling and warming without ice crystal growth and cell damage. Cold acclimation is not appropriate as pre-cultivation for potato plant (Hirai & Sakai, 1999; Schafer-Menuhr *et al*., 1996; Kaczmarczyk, 2008). The only method for potato vitrification is a water content decrease in samples, and than the rapid cooling and warming rate. Water content decrease is achieved by preculturing explants with osmotic compounds, air desiccation or vitrification. On bases, vitrification (Sarkar & Naik, 1998), droplet (Schafer-Menuhr *et al*., 1996) and recently vitrification-droplet (Halmagyi *et al*., 2004; Schafer-Menuhr *et al*., 1996) methods were developed or adapted for potato.

#### **3.3 Encapsulation-dehydration**

One of the other cryopreservation methods is encapsulation-dehydration. The shoot tips were encapsulated in an alginate gel. Experiments with dynamic dehydration studies demonstrated the necessity of meristems encapsulation (Benson *et al*., 1996; Grospietsch *et* 

Comparison of Cryopreservation Methods of

Cryoprotocol Steps The Procedure

Survival and regeneration evaluation

Table 1. Cryopreservation steps of garlic

minutes

temperature for 2 hours

seconds for thawing

Unripe bulbils dehydration

Cryopreservation

Thawing

Vegetatively Propagated Crops Based on Thermal Analysis 337

Fig. 2. The size and shape of shoot tips used for cryopreservation. (a) Garlic. Scale bar, 1 mm; (b) Potato. Scale bar, 0,25mm; (c) Hop. Scale bar, 0,25 mm and (d) Apple tree. Scale bar, 1 mm.

> Immersion in the loading solution (13,7 % (w/v) sucrose + 18,4 % (w/v) glycerol in the liquid medium) (Sakai *et al*., 1991) for 20

Dehydration by PVS3 (Nishizawa *et al*., 1993) at a laboratory

Aluminum foil stripes with 5-10 clusters of unripe bulbils plunged directly into liquid nitrogen at least for one hour in

Rapid warming immersion into a 40 °C water bath for 30-120

Sub-culture on MS medium supplemented with 0,2 mg L-1BAP and 0,02 NAA mg L-1 with 3 % sucrose for seven days in the dark

Removing and adding fresh PVS3 before freezing

Evaluation of survival after two weeks (Fig. 12a.) Evaluation of regeneration after 8-10 weeks

liquid nitrogen (Sarkar & Naik, 1998)

*al*., 1999; Hirai & Sakai, 1999). The encapsulation of shoot tips prolongs the dehydration period up to seven hours at a low relative humidity. The alginate beads without shoot tips had approximately the same dehydration-time curve. On the contrary no encapsulated shoot tips were completely dehydrated up to 1hour. The static dehydration of shoot tips was done over the various saturated salt solutions.
