**1. Introduction**

There is a trend to preserve the plant germplasm by not only conventional *ex situ* methods or *in vitro* techniques, but also, more recently, by cryopreservation. Cryopreservation techniques are based on the storage of plant samples at very low temperature at which practically no chemical reactions occur and consequently, neither aging nor genetic changes of plant material. There has been a great development progress of cryopreservation methods during last years. Cryopreservation becomes a highly utilized technique for germplasm conservation. Generally the cryopreservation is storage of the samples. The samples can be e.g. organs and shoots tips from *in vitro* culture, or from the field, such as mature, immature bulbils, cloves of garlic or dormant buds of fruit trees, at the ultra-low temperature (mainly –196 °C, the temperature of liquid nitrogen).

Although the technique was introduced for plants in the '70s, it has never been applied on a wide scale due to the high cost of cryo-freezers; indeed, it was used in order to escape the formation of lethal intracellular ice crystals, time-consuming and laborious slow-cooling procedures. A new cryogenic - vitrification technique is now available, aiming at the direct immersion of plant specimens from tissue cultures in liquid nitrogen, without resorting to an expensive apparatus for slow cooling and with a considerable simplification of the procedures (Benson, 2008). The vitrification method simplifies cryogenic process and makes possible an increased application of cryopreservation on wide-range plant genetic resources. The glassy state is the objective status of cryopreservation methods named vitrification.

The aim of this study is a comparison of different cryopreservation methods based on the vitrification achieved by dehydration and glass transition temperature (Tg), and their efficiency towards optimal regeneration of vegetatively propagated plants. The thermal characteristics, evaluation of frozen water content, and the glass transition temperature were measured by a differential scanning calorimeter.

#### **2. Importance of cryopreservation of vegetatively propagated plants**

Some of vegetatively propagated plants are not able to reproduce by seeds e.g. garlic plant (*Allium sativum* L.). The only way how to propagate it is to use its cloves or bulbils

Comparison of Cryopreservation Methods of

contamination or genetic alterations.

**3.2 Methods based on dehydration** 

**3.3 Encapsulation-dehydration** 

*et al*., 1996) methods were developed or adapted for potato.

**3.1 Cryoprotectants involved in vitrification method** 

necessary for the vitrification, which eliminates the formation of ice crystals.

Vegetatively Propagated Crops Based on Thermal Analysis 335

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

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

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

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* 

for seeding plants for further growing. The vegetatively propagated plant germplasm is endangered by abiotic and biotic factors in the field conditions. Although the production area of many vegetatively propagated plants has been decreasing, many local cultivars and varieties remain. In the presence of decreasing cultivar variability in production areas, diminishing of old orchards, as well as appearance of diseases close to field collection areas, the question of safely maintaining the broad genetic potential of fruit trees is arising.

Two safe methods ensure vegetatively propagated plant germplasm maintenance with a low risk of loss: slow-growth *in vitro* culture and the cryopreservation methods. Advantages of *in vitro* collection are aseptic and stable conditions of the cultivation and availability of the material during the year. A disadvantage is the necessity of sequential plant multiplication. Advantages of cryo-collection are low costs for its long-term maintenance and material stability. Disadvantages are a longer time for the plant to recover from stored material and a rather high input costs of the cryopreservation procedure. The best way how to maintain germplasm is the combination of both methods. The base collection should be maintained by *in vitro* collection that provides the material in case of requirements. Core collection of the most valuable material, should be backedup by cryo-collection for long-term storage, and plants are recovered just in case the genotype is lost from the base collection. For that reason, important vegetatively propagated plant collections have started to introduce accessions to slow-growth *in vitro* cultures and simultaneously in cryo-collection in liquid nitrogen (Gonzalez-Arnao *et al*., 2008; Keller *et al*., 2008; Kim *et al*., 2006).
