**6.3 Hop**

344 Current Frontiers in Cryopreservation

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

1.229J/(g·°C) -20.96°C

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





5.503J/g 1.643 % crystallized

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

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

to 30 °C by ramp temperature 10 °C min-1. Glass transitions were defined by the

for clarity according to crystallized water.






Heat Flow (W/g)

0.0

0.2

0.4

stability of material stored at ultra-low temperatures.





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).

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

Comparison of Cryopreservation Methods of

encapsulated shoot tips below 0,4 gH2O gDW-1.


along y-axis for clarity.

cryopreservation.



Heat Flow (W/g)


Vegetatively Propagated Crops Based on Thermal Analysis 347

was measured and no endothermal events representing water in ice crystal form were detected below 0,4 gH2O g DW-1. The value of 0,4 gH2O 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



0.24

Water content [g H O / g DM] <sup>2</sup>

0.26

0.39

0.39

0.72

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


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

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

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.

#### **6.4 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)



> -45.09°C(I) -0.06702W/g


0.4096J/(g·°C) -12.14°C

0.3043J/(g·°C) -9.54°C

104.7J/g 31.34 % crystallized



0

2

4

Heat Flow (mW)

6

8



Heat Flow (W/g)


171.6J/g 51.37 % crystallized

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


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

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)

rate was 10 °C min -1. Curves are shifted along y-axis for clarity.

**6.4 Apple tree** 

0.1

0.2

0.3

Heat Flow (W/g)

0.4

0.5

2000 for Windows (TA Instruments).

was measured and no endothermal events representing water in ice crystal form were detected below 0,4 gH2O g DW-1. The value of 0,4 gH2O 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 gH2O 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

Comparison of Cryopreservation Methods of

and (d) Apple tree. Scale bar, 5 mm.

average recovery in both crops.

at the CRI in Prague.

Vegetatively Propagated Crops Based on Thermal Analysis 349

Fig. 12. Plants regenerated into new plants after immersion in and thawing from the liquid nitrogen: (a) Garlic. Scale bar, 5 mm; (b) Potato. Scale bar, 2 mm; (c) Hop. Scale bar, 1 mm

The 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

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

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.

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 gH2O gDW-1. Water content decrease slows down markedly at the level of 0,6 gH2O 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.

#### **7. 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 accessions (the measurements on ripe bulbils were not presented, Grospietsch 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)

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 gH2O gDW-1. Water content decrease slows down markedly at the level of 0,6 gH2O 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

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

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 accessions (the measurements on ripe bulbils were not presented, Grospietsch 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-

frozen water; the less enthalpy the smaller amount of ice crystals in the sample.

**7. Regeneration of plants after cryopreservation** 

95% (Engelmann, 2011; Kim *et al*., 2006)

their survival at ultralow temperatures.

Fig. 12. Plants regenerated into new plants after immersion in and thawing from the liquid nitrogen: (a) Garlic. Scale bar, 5 mm; (b) Potato. Scale bar, 2 mm; (c) Hop. Scale bar, 1 mm and (d) Apple tree. Scale bar, 5 mm.

The 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

Survival [%]

Duncan's test) SD - standard deviation (P < 0,05).

to conserve a broader spectrum of apple tree germplasm.

**8. Comparison of different cryopreservation methods** 

Apple tree cultivar

Golden

regeneration).

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

SD

n

Number of freezing

SD Regrowth [%]

Alkmene 6 a 0,4 6 a 0,4 32 2

Delicious 75 bc 27,9 55 bcd 24,7 39 3 Greensleeves 75 bc 15,0 53 bcd 7,5 30 2 Chodské 63 bc 17,6 46 abcd 13,8 84 5 Idared 34 abc 13,5 34 abc 13,5 45 2 Jonagold 63 bc 13,4 44 abcd 26,2 50 3 McIntosh 85 c 15,0 85 d 15,0 20 2 Prima 28 ab 15,0 21 ab 8,0 20 2 Rubín 78 bc 11,0 78 cd 11,0 32 2 Zvonkové 44 abc 18,7 4 a 5,4 56 4 Average 75 21,7 43 21,2 41 3

a-d average followed by the same index did not significantly differ at P<0,05 (analysis of variance –

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

*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

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

the needs for successful cryopreservation of apple tree *in vitro* germplasm.

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.

**Garlic**

 ripe bulbils unripe bulbils

0 10 20 30 40 50 60 70 80 90

**Potato**

0 10 20 30 40 50 60 70 80 90 100

**Hop**

Regeneration frequency (accession)

Regeneration frequency (accession)

Regeneration frequency (accession)

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

0 10 20 30 40 50 60 70 80 90 100 Regeneration after cryopreservation (%)

hop.



a-d average followed by the same index did not significantly differ at P<0,05 (analysis of variance – Duncan's test) SD - standard deviation (P < 0,05).

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).

*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.
