**6. Water content and glass transition**

#### **6.1 Garlic**

340 Current Frontiers in Cryopreservation

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

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



Fig. 3. Example of apple tree shoot tips heat flow response to the temperature. Cooling and




Exotherm

Endotherm

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

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

samples is considered a critical point of plant survival at ultra-low temperatures.

cooling/heating rate of the sample is 10 °C min-1.

Glass transition


warming rate was 10 °C min -1. Exo up.





Heat Flow (W/g)

0.0

0.2

0.4

either nitrogen or helium.

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 with decreases of water after dehydration.

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.

Comparison of Cryopreservation Methods of

observed after 0,75 M sucrose treatment.

was determined by the DSC analysis.

0

1

2

3

Water content (g H O gDW

 2


4

5

6

content.

Vegetatively Propagated Crops Based on Thermal Analysis 343

(2004). Halmagyi *et al*., (2004) showed the highest plant regeneration after cryopreservation following a pre-treatment with 0,5 M sucrose. Similarly, Sarkar and Naik (1998) found a slightly negative effect of 0,7 M sucrose pre-treatment compared in comparison with 0,5 M or 0,3 M sucrose pre-treatment. In the present study the injury of potato explants was not

Total water content in the shoot tips after nodal cutting pre-culture was approximately 5 g of H2O per 1 g of dry mass (gH2O g DW-1) (Fig. 6.). Frozen water content in shoot tips was 4,3 gH2O g DW-1 and the unfrozen 0,7 gH2O g DW-1. Subsequently shoot tips were isolated and loaded with 0,7 M sucrose in a Petri dish on filter paper for overnight. Total water content of shoot tips decreased to 2,1 gH2O g DW-1, from which 1,4 gH2O g DW-1 represents the frozen water fraction and 0,7 gH2O g DW-1 the unfrozen water fraction. Because the total water content and frozen water fraction decreased but the unfrozen fraction did not change, the ratio of frozen/unfrozen water content (WCf/WCu) decreased from 6 to 1,9. The following air dehydration resulted in a decrease of total water content due to both water fractions decrease. After 1,5h air dehydration above silicagel the total water content in shoot tips was 0,49 gH2O g DW-1 from which the frozen water content was 0,09 gH2O g DW-1, and the unfrozen 0,4 gH2O g DW-1. Resulting WCf/WCu ratio decreased to 0,22. The prolonged dehydration decreased both water fractions. After 2h air dehydration above silicagel the total water content in shoot tips was 0,28 gH2O g DW-1 from which 0,006 gH2O g DW-1 belonged to the frozen fraction and 0,276 gH2O g DW-1 to the unfrozen fraction. The WCf/WCu ratio decreased to 0,12 after 2h air dehydration of shoot tips above silicagel, which represents 2 % crystallized water of the total water

Fig. 6. The progress of dehydration of potato explants (cv. Désirée) after specific steps of cryoprotocol. Explants were pre-cultured on medium with 0,7 M sucrose. The isolated shoot tips were loaded with 0,7 M sucrose solution for overnight. The loaded shoot tips were dehydrated by dry air above silicagel for 2 hours. The amount of frozen and unfrozen water

precultured explants loaded shoot tips 1,5h air dehydration -> -> 2h air dehydration Progress of dehydration

frozen water unfrozen water

Fig. 5. Glass transition temperature of *Allium* shoot tips after moisture loss by dehydration in the Plant Vitrification Solution 3 (PVS3). Circles show the glass transition midpoint in shoot tips and bars show the onset and endset of glass transition. Squares show the glass transition of PVS3 in shoots. The full line is for the glass transition of PVS3 without shoots. The dashed line above is the endset of glass transition and below the onset of glass transition for PVS3.

Glass transition of garlic shoot tips was measured after different times of treatment – unripe bulbils in PVS3 at 23 °C (Fig. 5). At each curve, there were two S-shape heat flow changes during warming of the samples, typical for glass transition. The lower glass transition temperature on unripe bulbils heat flow curves coincides within the range of onset and endset of the glass transition temperature of PVS3 measured after unloading unripe bulbils. This glass transition temperature can be of PVS3 coating on the surface of the shoot tips immersed in PVS3.

The high glass transition temperature corresponds to glass transition of the shoot tips because at this range of temperature there were no thermal events on the PVS3 temperature dependent curve. There is no significant difference in the change of shoot tip glass transition changes from 0,5 to 2,5 hours of PVS3 treatment. The detectable glass transition was found between -30 °C and -39 °C. The average glass transition temperature is -33,5 °C after 0,5 hour. From these results it is obvious that the glass transition at higher temperature is for shoot tips saturated with PVS3. So, for the survival of shoot tips after thawing from liquid nitrogen, the second glass transition which occurs at higher temperatures is important (Zamecnik *et al.*, 2011).

#### **6.2 Potato**

Nodal cuttings were pre-cultured on medium with added sucrose solution. The final sucrose concentration in medium was 0,7 M. The importance of sucrose pre-treatment before potato cryopreservation proved by Grospietsch *et al*., (1999) and Halmagyi *et al*.,

Treatment time (hours)

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

PVS3

Shoots

Fig. 5. Glass transition temperature of *Allium* shoot tips after moisture loss by dehydration in the Plant Vitrification Solution 3 (PVS3). Circles show the glass transition midpoint in shoot tips and bars show the onset and endset of glass transition. Squares show the glass transition of PVS3 in shoots. The full line is for the glass transition of PVS3 without shoots.

Glass transition of garlic shoot tips was measured after different times of treatment – unripe bulbils in PVS3 at 23 °C (Fig. 5). At each curve, there were two S-shape heat flow changes during warming of the samples, typical for glass transition. The lower glass transition temperature on unripe bulbils heat flow curves coincides within the range of onset and endset of the glass transition temperature of PVS3 measured after unloading unripe bulbils. This glass transition temperature can be of PVS3 coating on the surface of the shoot tips

The high glass transition temperature corresponds to glass transition of the shoot tips because at this range of temperature there were no thermal events on the PVS3 temperature dependent curve. There is no significant difference in the change of shoot tip glass transition changes from 0,5 to 2,5 hours of PVS3 treatment. The detectable glass transition was found between -30 °C and -39 °C. The average glass transition temperature is -33,5 °C after 0,5 hour. From these results it is obvious that the glass transition at higher temperature is for shoot tips saturated with PVS3. So, for the survival of shoot tips after thawing from liquid nitrogen, the second glass transition which occurs at higher

Nodal cuttings were pre-cultured on medium with added sucrose solution. The final sucrose concentration in medium was 0,7 M. The importance of sucrose pre-treatment before potato cryopreservation proved by Grospietsch *et al*., (1999) and Halmagyi *et al*.,

The dashed line above is the endset of glass transition and below the onset of glass

transition for PVS3.




Glass transition temperature (°

C




immersed in PVS3.

**6.2 Potato** 

temperatures is important (Zamecnik *et al.*, 2011).

(2004). Halmagyi *et al*., (2004) showed the highest plant regeneration after cryopreservation following a pre-treatment with 0,5 M sucrose. Similarly, Sarkar and Naik (1998) found a slightly negative effect of 0,7 M sucrose pre-treatment compared in comparison with 0,5 M or 0,3 M sucrose pre-treatment. In the present study the injury of potato explants was not observed after 0,75 M sucrose treatment.

Total water content in the shoot tips after nodal cutting pre-culture was approximately 5 g of H2O per 1 g of dry mass (gH2O g DW-1) (Fig. 6.). Frozen water content in shoot tips was 4,3 gH2O g DW-1 and the unfrozen 0,7 gH2O g DW-1. Subsequently shoot tips were isolated and loaded with 0,7 M sucrose in a Petri dish on filter paper for overnight. Total water content of shoot tips decreased to 2,1 gH2O g DW-1, from which 1,4 gH2O g DW-1 represents the frozen water fraction and 0,7 gH2O g DW-1 the unfrozen water fraction. Because the total water content and frozen water fraction decreased but the unfrozen fraction did not change, the ratio of frozen/unfrozen water content (WCf/WCu) decreased from 6 to 1,9. The following air dehydration resulted in a decrease of total water content due to both water fractions decrease. After 1,5h air dehydration above silicagel the total water content in shoot tips was 0,49 gH2O g DW-1 from which the frozen water content was 0,09 gH2O g DW-1, and the unfrozen 0,4 gH2O g DW-1. Resulting WCf/WCu ratio decreased to 0,22. The prolonged dehydration decreased both water fractions. After 2h air dehydration above silicagel the total water content in shoot tips was 0,28 gH2O g DW-1 from which 0,006 gH2O g DW-1 belonged to the frozen fraction and 0,276 gH2O g DW-1 to the unfrozen fraction. The WCf/WCu ratio decreased to 0,12 after 2h air dehydration of shoot tips above silicagel, which represents 2 % crystallized water of the total water content.

Fig. 6. The progress of dehydration of potato explants (cv. Désirée) after specific steps of cryoprotocol. Explants were pre-cultured on medium with 0,7 M sucrose. The isolated shoot tips were loaded with 0,7 M sucrose solution for overnight. The loaded shoot tips were dehydrated by dry air above silicagel for 2 hours. The amount of frozen and unfrozen water was determined by the DSC analysis.

Comparison of Cryopreservation Methods of

**6.3 Hop** 

water content (Fig. 9).

dehydration (cv. Saazer).

Survival, Regeneration (%)

Vegetatively Propagated Crops Based on Thermal Analysis 345

osmotic pre-treatment, shoot tips sucrose loading and their air dehydration on aluminum foils was used for storage of 58 selected potato. All plant accessions prepared for storage in cryo-bank were virus-free. Average post-thaw recovery of hop and potato was 36 % and 25

Isolated hop shoot tips (cv. Saazer) were dehydrated by air above silicagel (Fig. 8). Water content was 2,4 g water per 1g dry mass before air dehydration. The highest water decrease was measured during the first 30 minutes of dehydration. Water content of hop shoot tips was 0,68 gH2O gDW-1 after 32 minutes of dehydration. Shoot tips water content decreased below 0,5 gH2O gDW-1 after 70 minutes of dehydration and reached 0,4 gH2O gDW-1 after 100 minutes of dehydration. After 120 minutes the shoot tips water content was 0,37 gH2O g DW-1. The plant regeneration depended on the time of dehydration, which was influenced by the shoot tips water content. The highest explant regeneration was achieved after 90

In a former study, a decrease in the endothermic peak was found during air dehydration by encapsulation-dehydration method used for hop cryopreservation (Martinez *et al*.,1998; Martinez *et al*., 1999; Martinez & Revilla, 1998). A negligible amount of freezable water was detected in shoot tips after the water content decreased to 18 % and no freezable water was found at a water content of 14 %. The glass transition temperature was found at a water content of 18 % and lower. The temperature of glass transition increased with a decrease of

Fig. 8. Survival (empty circles) and regeneration (full circles) of hop explants during

70 75 80 85 90 95 100 105 110 Dehydration time (min)

%, respectively. Recovery of new plants was successful in all tested genotypes.

minutes of dehydration at a water content close to 0,4 gH2O g DW-1.

The decrease in percentage of crystallized water in shoot tips during 1,75 to 2h air dehydration is illustrated in Fig. 6. The crystallized water content decreased from approximately 9% to 2%. Dehydration of shoot tips was connected to the glass transition temperature increase from -38 to -32 °C. The optimal water content of potato shoot tips was approximately 0,4 gH2O g DW-1 that was obtained between 1,5h and 2h air dehydration above silicagel according to the size of particular genotype shoot tips. The temperature of glass transition was approximately -35 °C and the amount of frozen water was very small but still detectable (Fig. 7). Decrease in water content and onset of melting temperature was also found after dehydration by PVS2 solution or 10 % DMSO (Kaczmarczyk, 2008, Kaczmarczyk *et al.* 2011). However the Tg found by these cryoprotectants was lower than - 100 °C. The higher temperature of glass transition found in this study indicated a higher stability of material stored at ultra-low temperatures.

Fig. 7. DSC curves of air dehydrated potato shoot tips (cv. Désirée) air-dehydrated above silicagel for 1,75 to 2 hours. Heat flow was evaluated during warming the samples from -130 to 30 °C by ramp temperature 10 °C min-1. Glass transitions were defined by the temperature of glass transition, change of heat flow per g of sample and change in specific heat capacity (Cp). Melting exotherms are defined by the onset temperature of melting, enthalpy change of thermal event, and crystallinity of water. Curves are shifted along y-axis for clarity according to crystallized water.

The most valuable accessions from sub-collection of old potato cultivars of the Czech origin were selected from the potato in vitro-bank at the Crop Research Institute (CRI) to store them by cryopreservation method. A new cryopreservation method based on nodal cutting osmotic pre-treatment, shoot tips sucrose loading and their air dehydration on aluminum foils was used for storage of 58 selected potato. All plant accessions prepared for storage in cryo-bank were virus-free. Average post-thaw recovery of hop and potato was 36 % and 25 %, respectively. Recovery of new plants was successful in all tested genotypes.
