**5. Thermal analysis**

Differential scanning calorimetry (DSC) belongs to thermal methods that can be used for measurement and determination of phase and glass transitions for cryopreservation. In principle, the DSC measures the temperatures and heat flows associated with transitions in

Comparison of Cryopreservation Methods of

Windows (TA Instruments).

**6.1 Garlic** 

**6. Water content and glass transition** 

with decreases of water after dehydration.

0

0,5

1

1,5

Water content (gH O gFW

2

2

2,5


3

3,5

2009; Šesták & Zámečník, 2007; Zámečník *et al*., 2007)

Vegetatively Propagated Crops Based on Thermal Analysis 341

properties before, during and after cryopreservation (Benson *et al*., 1996; Faltus & Zámečník,

There is an example (Fig. 3) of measured thermal characteristics of shoot tips of apple tree *in vitro* culture cv. Greensleeves by the DSC. Samples of an approximate weight of 10 mg were crimped in an aluminium sample pan and cooled from room temperature to -120 °C. Cooling and heating rate was 10 °C min-1. The glass transition, exothermic and endothermic characteristics were analyzed in detail during heating. Thermal characteristics were measured by DSC TA 2920 (TA Instruments) and evaluated by Universal Analysis 2000 for

Water content during dehydration of garlic cv. Djambul 2 clusters of shoot tips by PVS3 (Fig. 4). Total amount of water (solid line) and amount of crystallized water (dash line) in shoot tips treated with PVS3 rapidly descend during the first 1,5 hours and further is constant. Crystallized water reaches minimum after 1,75 hours of PVS3 treatment. In this case the decrease of water content in the unripe bulbils is probably so low that it can have no further influence on the glass transition change. In comparison with the measurements in this study on the apple tree shoot tips (see below), the glass transition temperature increases

Fig. 4. Unripe garlic bulbils water content (empty circle) and the part of frozen water (full circles) during PVS3 treatment. Note: The unripe bulbils were in the loading solution first 20 minutes than they were immersed in to the PVS3. The bars are standard deviation of mean.

0 0,5 1 1,5 2 2,5 3

Treatment time (hours)

plant material as a function of time and temperature. It gives information about endothermic or exothermic changes or changes in heat capacity. The obtained data can be used for determination of glass transition, temperature of ice nucleation, melting, boiling, crystallization time and kinetic reaction – the most important characteristics useful for cryopreservation (Zámečník & Faltus, 2009). The danger of nucleation and subsequent intracellular ice crystallization leading to frost damage during cooling and rewarming of the samples is considered a critical point of plant survival at ultra-low temperatures.

The differential scanning calorimetry method is based on the regulated decrease/increase temperature of the sample and reference and the measurement of temperature and heat flow corresponding to the sample. There are two different types of the differential scanning calorimeters. The power compensation DSC type directly measures heat release/uptake from the sample and the heat flow type measures differences of temperature between reference and sample and recalculates the differential heat flux. The most common cooling/heating rate of the sample is 10 °C min-1.

Fig. 3. Example of apple tree shoot tips heat flow response to the temperature. Cooling and warming rate was 10 °C min -1. Exo up.

In our experiments we used shoot tips of *in vitro* cultures of apple tree. Samples with different water content were obtained by air dehydration of alginate encapsulated shoot tips in the flow box or by dehydration at 4 °C of *in vitro* cultures. For DSC measurement dissected shoot tips were placed in aluminum sample pans and measured by Differential Scanning Calorimeter TA2920. Samples were cooled down to -120 °C (rate of 10 °C min-1). The data were collected during heating to 20 °C (rate of 10 °C min-1). The purge gas was either nitrogen or helium.

A Differential Scanning Calorimeter is used as a main tool in cryobiology to assist cryopreservation protocol development, to store thermograms as a documentation of cryoprotocols in the use and to keep information about stored samples and their thermal properties before, during and after cryopreservation (Benson *et al*., 1996; Faltus & Zámečník, 2009; Šesták & Zámečník, 2007; Zámečník *et al*., 2007)

There is an example (Fig. 3) of measured thermal characteristics of shoot tips of apple tree *in vitro* culture cv. Greensleeves by the DSC. Samples of an approximate weight of 10 mg were crimped in an aluminium sample pan and cooled from room temperature to -120 °C. Cooling and heating rate was 10 °C min-1. The glass transition, exothermic and endothermic characteristics were analyzed in detail during heating. Thermal characteristics were measured by DSC TA 2920 (TA Instruments) and evaluated by Universal Analysis 2000 for Windows (TA Instruments).
