Comparison of Cryopreservation Methods of Vegetatively Propagated Crops Based on Thermal Analysis

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).


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 (T g ), 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.
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).

Cryopreservation methods
The latest results from the field of low temperature biology suggest that the main factor influencing the success of the cryopreservation method is the maintenance of a glassy state in plant samples and the avoidance of ice nucleation.The danger of ice nucleation and subsequent ice crystallization leading to frost damage during cooling and rewarming of samples is considered as a critical point of cryopreservation.That is the reason why many of the new progressive methods use and involve a glassy state in plant material intended for cryopreservation.Knowledge of the glass transition temperature is useful not only for improving methods involving glassy state in plant shoots tips.It also provides information essential for the long-term storage of shoot tips.This biotechnology is based on the induction of the vitrification status -glass induction by dehydration, addition of cryoprotectants and a very fast decrease in temperature.Vitrification can be achieved in a number of ways (Sakai & Engelmann, 2007) but they usually all have the results of increased solute concentration to a critical viscosity.Low water content minimizes the ice crystallization that is potentially dangerous for plant cells and increases the temperature of glass transition.Supposing that the change of water status in the certain range is not limiting for plant regeneration.Plant Vitrification Solutions (PVS) marked with numbers according to the specific mixture of basic cryoprotectants and their concentrations are usually used for osmotic dehydration.Another cryopreservation method used, is based on desiccation in the air-flow cabinet.It is defined with the flow rate, temperature and humidity or on desiccation over various saturated salt solutions with steady-state activity of water.
Comparison of Cryopreservation Methods of Vegetatively Propagated Crops Based on Thermal Analysis 335

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 t h e y c a n b e s t o r e d f o r a n i n d e f i n i t e p e r i o d of time without undergoing the risks of contamination or genetic alterations.

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.

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 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.

Garlic
The unripe topsets were surface sterilized by chloramines and 75% ethanol and from this point, all preparations were performed under sterile conditions using sterile instruments and culture media in a laminar flow box.Opening the surface sterilized topset in sterile condition, all the inside structures were sterile (Fig. 1a).The sterile unripe bulblets were removed and cuts were made to the clusters or clumps of 3-8 bulbils.The bulbils varied in the thickness (approximately 2 mm) and in the length (3-5 mm) depending on the genotype and stage of ripening.Inside the unripe bulbils shoot tips were (Fig. 2a) with meristematic tissue.
Pre-culture of unripe clusters of bulbils was done on the MS culture medium (Murashige & Skoog, 1962) with 0,2 mg L -1 BAP and 0,02 NAA mg L -1 with 10 % sucrose for 20-24 h at 22 °C and 16 h light in Petri dishes sealed with Parafilm.

Cryoprotocol Steps
The Procedure Unripe bulbils dehydration 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 minutes Cryopreservation Dehydration by PVS3 (Nishizawa et al., 1993) at a laboratory temperature for 2 hours Removing and adding fresh PVS3 before freezing Aluminum foil stripes with 5-10 clusters of unripe bulbils plunged directly into liquid nitrogen at least for one hour in liquid nitrogen (Sarkar & Naik, 1998) Thawing Rapid warming immersion into a 40 °C water bath for 30-120 seconds for thawing Survival and regeneration evaluation Sub-culture on MS medium supplemented with 0,2 mg L -1 BAP and 0,02 NAA mg L -1 with 3 % sucrose for seven days in the dark Evaluation of survival after two weeks (Fig. 12a.)Evaluation of regeneration after 8-10 weeks Table 1.Cryopreservation steps of garlic www.intechopen.com
Nodal cuttings were planted in the same conditions as the pre-cultured plants but only 50 ml medium was used per one box.After 4 day pre-culture, lateral buds elongated to at least 1 mm (Fig. 2b).Subsequently, 25 ml 2 M sucrose was added into each container and explants were cultivated at the same conditions for the next 5-6 days.
The second day the shoot tips were transferred (20 tips per foil) onto aluminum foils (20 x 6 x 0.05 mm) Dehydration above silicagel for 1,75 -2 h.

Cryopreservation
Plunging aluminum foils into liquid nitrogen Stored in cryovials (two foils with 20 shoot tips per vial).

Thawing
Alluminum foils plunged rapidly into the water bath at a laboratory temperature.
Transfer immediately onto the recovery medium (Grospietsch et al., 1999) with the same composition as the pre-culture medium but with phytohormones (0,5 mg L -1 IAA + 0,.5 mg L -1 Kinetin + 0,2 mg L -1 GA3) Survival and regeneration evaluation Survival was defined by shoot tip growth and by green color and recovery as a new explant development (Fig. 12b.)Plant regeneration was evaluated 2 and 8 weeks after cryopreservation.
Table 2. Cryopreservation steps of potato
Nodal cuttings were planted in the same conditions as during explant multiplication but only 50 ml medium was used per one box (Fig 1c).After 7-10 d pre-culture, lateral buds elongated to 1-2 mm.Then the explants were transferred into cold acclimation conditions at 4 °C for 7-10 days.Subsequently 25 ml 0,7 M sucrose was added into each container and explants (Fig. 1c.) were cultivated at the same conditions for the next 7-10 days.
Cryopreservation steps for hop shoot tips were the same as for potato (see Tab. 2).

Cryoprotocol Steps
The Procedure

Shoot tips dehydration
One to two week subcultivation on fresh MS medium, Cold hardening for 4-6 weeks at +4± 1 °C, short photoperiod (8/16, L/D) of light intensity 25 µmol m -2 s -1 Petri dish with 16 ml of MS medium and poured with 16 ml of 2M sucrose solution for 48 hours Table 3. Cryopreservation steps of apple tree

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 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).

Water content and glass transition 6.1 Garlic
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.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).

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) (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 H 2 O per 1 g of dry mass (gH 2 O g DW -1 ) (Fig. 6.).Frozen water content in shoot tips was 4,3 gH 2 O g DW -1 and the unfrozen 0,7 gH 2 O 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 gH 2 O g DW -1 , from which 1,4 gH 2 O g DW -1 represents the frozen water fraction and 0,7 gH 2 O 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 (WC f /WC u ) 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 gH 2 O g DW -1 from which the frozen water content was 0,09 gH 2 O g DW -1 , and the unfrozen 0,4 gH 2 O 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 gH 2 O g DW -1 from which 0,006 gH 2 O g DW -1 belonged to the frozen fraction and 0,276 gH 2 O g DW -1 to the unfrozen fraction.The WC f /WC u 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.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 gH 2 O 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 T g 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.

Temperature (°C)
Exo Up Universal V4.7A TA 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.

Hop
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 gH 2 O gDW -1 after 32 minutes of dehydration.Shoot tips water content decreased below 0,5 gH 2 O gDW -1 after 70 minutes of dehydration and reached 0,4 gH 2 O gDW -1 after 100 minutes of dehydration.After 120 minutes the shoot tips water content was 0,37 gH 2 O 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 minutes of dehydration at a water content close to 0,4 gH 2 O g DW -1 .
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 water content (Fig. 9).Dehydration time (min) Survival, Regeneration (%) Fig. 9. Heat flow response to the temperature of hop shoot tips.Glass transition temperature of hop shoot tips after moisture loss by dehydration above silicagel.Cooling and warming rate was 10 °C min -1 .Curves are shifted along y-axis for clarity.

Apple tree
In Fig. 10, there is an example of measured thermal characteristics of encapsulated shoot tips of apple tree in vitro culture cv.Greensleeves by the DSC.Samples of approximate weight of 10 mg were crimped in an aluminium sample pan and cooled from room temperature to -120 °C.The cooling and heating rate was 10 °C min -1 .The glass transition, exotherm and endotherm characteristics were analysed in detail during heating.Thermal characteristics were measured by DSC TA 2920 (TA Instruments) and evaluated by Universal Analysis 2000 for Windows (TA Instruments).
The course of dehydration of encapsulated shoot tips of in vitro cultures of apple tree cv.
Greensleeves in an open Petri dish exposed to air flow in laminar flow hood at laboratory temperature is demonstrated in Fig. 10.The determination of water content of 20 encapsulated shoot tips placed in the Petri dish was done by weighing the shoot tips during dehydration after approximately 4 hours of drying to the constant weight in an oven (105 °C).The water content was calculated as a proportion of g of water to g of dry matter.From measurement it was evident, that the dehydration consists of two parts; a faster one at the beginning and a slower one after approximately 2 hours of dehydration.From 2 h of dehydration, the level of water content in encapsulated apple tree shoot tips is almost constant.In Fig. 10, there are thermal characteristics (during heating) of encapsulated apple tree shoot tips during their dehydration in the air flow in flow hood.The more dehydrated the samples, the higher the glass transition temperature (characterised by the inflex point I) -37.13°C(I) 0.06740W/g 0.4092J/(g

347
was measured and no endothermal events representing water in ice crystal form were detected below 0,4 gH 2 O g DW -1 .The value of 0,4 gH 2 O g DW -1 dehydration level corresponds to the levels recommended also by other authors (Gupta & Reed, 2006;Martinez et al., 1999;Wu et al., 1999).The integration of endotherm areas of shoot tip and alginate confirms the importance of dehydration to the levels when ice crystals are not present in shoot tip tissues (Figs. 10,11).The energy counted as integration of the endothermic peak corresponded to the amount of frozen water; the less energy, the smaller amount of ice crystals in the sample.These thermal results led us to dehydrating encapsulated shoot tips below 0,4 gH 2 O gDW -1 .Fig. 10.Glass transition temperature as an inflection point of heat flow change of encapsulated apple tree shoot tips after water loss expressed as final water content (figures behind the end of the separate curves) by dehydration in the air flow.Curves are shifted along y-axis for clarity.
Dehydration curves corresponding to the loss of water from encapsulated in vitro shoot tips were measured (Figs. 10,11).During the dehydration procedure of cryopreservation The proper time/level of dehydration must be taken into consideration for successful cryopreservation.
The less water in plant tissues, the less probable damage from ice crystal formation and growth.On the other side plant tissues withstand only certain dehydration.The most appropriate level of dehydration is determined by DSC by measurement of frozen and unfrozen water (generally it is possible to say water in glassy state) (Fig. 11).After 4h dehydration of encapsulated in vitro shoot tips the water content decreased below 0,3 gH 2 O gDW -1 .Water content decrease slows down markedly at the level of 0,6 gH 2 O gDW -1 .From this level of dehydration, both exotherms and endotherms start to disappear which corresponded to the end of ice crystal formation and the start of glass transitions with high change of heat capacity (Fig. 11).The survival and regeneration of cryopreserved apple tree shoot tips, cultivar Greensleeves, were 75 % and 53 % respectively after 4h dehydration.Non-dehydrated shoot tips neither survived nor regenerated.Dehydration of shoot tips to the level of glass formation is a crucial factor for their survival at ultralow temperatures.
Fig. 11.The amount of frozen water of apple tree shoot tips and alginate beads during dehydration was as the integration of endotherm areas of shoot tip and alginate.Samples of an approximate weight of 3-10 mg were crimped in an aluminum sample pan and cooled from room temperature to -120 °C.The cooling and heating rate was 10 °C min -1 .The enthalpy counted as an integration of the endothermic peak corresponded to the amount of frozen water; the less enthalpy the smaller amount of ice crystals in the sample.

Regeneration of plants after cryopreservation
The regeneration rate of unripe garlic bulbils was close to 100 % in comparison with the lower regeneration rate of ripe bulbils.The results for ripe bulbils were done on 173 a c c e s s i o n s ( t h e m e a s u r e m e n t s o n r i p e b u l b i l s w e r e n o t p r e s e n t e d , G r o s p i e t s c h unpublished).The average regeneration rate of ripe bulbils was 40 % and unripe bulbils near 100%.The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of garlic varieties also with high regeneration percentages ranging between 77,4-95% (Engelmann, 2011;Kim et al., 2006) www.intechopen.comThe average recovery after cryopreservation of fifty potato cultivars was 24,8% and average hop recovery was 30,5%.Plant recovery eas improved due to the cryoprotocol and media modifications and the average recovery of potato and hop in the year 2007 was 29,1% and 35,5%, respectively (Fig. 13).The highest frequency of plant recovery was near to the average recovery in both crops.
To improve the stability and safety of potato collection, the cryo-collection of the Czech potato germplasm was established.The sub-collection of old potato varieties of the Czech origin was selected as the most important part of potato germplasm kept in the Czech In Vitro Bank of Potato.Fifty eight selected genotypes were cryopreserved by a new method based on osmotic adjustment of explants with sucrose and following air-dehydration.Currently these 58 old potato cultivars of the Czech origin were backed up in cryo-collection at the CRI in Prague.
The differences in plant survival and regeneration exist either among species or cultivars.Example of survival and regeneration of apple tree in vitro cultures cryopreserved by the encapsulation-dehydration method are shown in (Tab.4).The differences can be caused either by different reaction of cultivars in cryopreservation protocol or cold hardening conditions or in vitro cultivation.For example, the cultivar McIntosh belongs to very cold resistant cultivars and also has very high regeneration after cryopreservation.On the contrary, the very cold tender cultivar Zvonkové had no survival in laboratory frost tolerance test on dormant buds (data not shown) but in vitro cultures were able to survive the cryopreservation procedure, although with very weak regeneration.

Comparison of Cryopreservation Methods of Vegetatively Propagated Crops Based on Thermal Analysis 351
There is clear evidence for the necessity of physiological and biochemical adaptations of cryopreservation procedures according to the different demands of used cultivars to fulfill the needs for successful cryopreservation of apple tree in vitro germplasm.In vitro shoot tips of apple tree were cryopreserved also by vitrification (PVS2) and preculture dehydration methods, but the results were not adequate (Tab.5).Thus the basic approach of cryopreservation of fruit trees in our laboratory was the encapsulationdehydration method.The average survival and regeneration of evaluated apple tree cultivars were 75 % and 53 %, respectively (Tab.4).Similar values of regeneration were obtained by (Condello et al., 2011) with the droplet-vitrification method in two apple tree cultivars.On the other hand, (Wu et al., 2001) reached regeneration of up to 86 %.They recommended a prolonged subcultivation of mother plants and their cold acclimation, which decreased water content in shoot tips and subsequently increased the regeneration in all cryopreservation procedures they evaluated.According to our unpublished data and in concordance with other authors, (Wu et al., 2001;Condello et al., 2011), the adaptation of the in vitro mother plants appears to be one of the important steps for improving regeneration of cryopreserved cultivars with lower survival.The encapsulation-dehydration cryopreservation method is a suitable tool to conserve a broader spectrum of apple tree germplasm.

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 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 (T g ) -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).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.

Conclusion
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
Encapsulation of shoot tips (Fig.2d. ) with meristematic cells in 3% w/v dipped into alginate in 0,75M sucrose for 10 min and then drop into 0,1 M CaCl2 in 0,6 M sucrose for 10 min to net the alginate and form a bead Cryopreservation Beads are gently dried on a sterile filter paper Additional dehydration in the laminar flow box at laboratory temperature and dehydrated for different time up to 4 -5 hours, Placed in 2 ml cryovials and plunged in liquid nitrogen.Thawing Shoot tips with beads were warmed up by plunging cryovials in 40 °C water Survival and regeneration evaluation Survival was defined by shoot tip growth and by green color and regenerated as a new explant development (Fig. 12d.)

Fig. 4 .
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.

Fig. 5 .
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.
and Halmagyi et al.,

Fig. 7 .
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 (C p ). 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.

Fig. 13 .
Fig. 13.Regeneration frequency of genotypes (accessions) of garlic, potato and hop stored in the Cryobank of vegetatively propagated crops.Altogether, 129 accessions were evaluated in ripe garlic bulbils, 34 accessions in unripe garlic bulbils, 58 accessions in potato and 50 in hop.

Table 4 .
Survival and regrowth of apple tree cultivars after encapsulation-dehydration cryopreservation protocol.(n = total number of shoot tips used for evaluations of regeneration).