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1. 95
CryoLetters 29(2), 95-110 (2008)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
APPLICATIONS OF DIFFERENTIAL SCANNING CALORIMETRY IN
DEVELOPING CRYOPRESERVATION STRATEGIES FOR Parkia
speciosa, A TROPICAL TREE PRODUCING RECALCITRANT SEEDS
Jayanthi Nadarajan12*a
, Marzalina Mansor2
, Baskaran Krishnapillay2
, Harry J. Staines1
Erica E. Benson3
and Keith Harding3
1
University of Abertay Dundee, Bell Street. Dundee DD1 1HG, Scotland, UK
2
Forest Research Institute of Malaysia (FRIM), Kepong, 52109 Kuala Lumpur, Malaysia
3
Research Scientists, Conservation, Environmental Science and Biotechnology, Damar, Drum
Road, Cupar Muir, Fife, KY15 5RJ, Scotland, UK
*Author for correspondence (email: j.nadarajan@kew.org)
a
(Current contact address) Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West
Sussex RH17 6TN, UK
Abstract
Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree
withstood cryopreservation using encapsulation-vitrification in combination with trehalose
preculture. Differential Scanning Calorimetry (DSC) revealed that trehalose moderated the
thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass
transition temperature (Tg) (-50.21 ± 1.07ºC) when applied in combination with 5% (w/v)
trehalose. The Tg increased to -40.22 ± 0.95ºC as the sugar’s concentration was decreased to
2.5% (w/v). Tg heat capacity for shoot-tips treated with 2.5% and 5% (w/v) trehalose and
exposed to PVS2 for 30 min increased from 0.17 ± 0.05 to 0.23 ± 0.01 J.g-1
respectively.
Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30
min PVS2 treatment, whereas the melt enthalpy for control shoots was >150 J.g-1
and
decreased to ca. 60 J.g-1
with 2.5% (w/v) trehalose. For 5% and 10% (w/v) trehalose
treatments, enthalpy declined to ca. 24 and 12 J.g-1
respectively and freezing points were
depressed to –75ºC and –85ºC with 2.5% and 5% trehalose (w/v), respectively. DSC
elucidated the critical points at which vitrification occurred in germplasm exposed to
trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70% survival was found
optimal for stable glass formation during cooling and on rewarming.
Keywords: Parkia speciosa, differential scanning calorimetry, vitrification, trehalose
INTRODUCTION
Orthodox and intermediate seeds can be cryopreservation tolerant, however this is not the
case for recalcitrant seeds which have many storage problems. The development of cryo-
conservation for sensitive germplasm requires the consideration of both cryogenic
(cryoprotectant and low temperature) and non-cryogenic (pre- and post-storage culture)
factors. It is also important to study the physiology of recalcitrant germplasm as it often has
greater sensitivity to cryoinjury, osmotic stress, desiccation and freezing. Recalcitrant seeds

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are known to be bulky with fleshy cotyledons; they are metabolically active and highly
hydrated making cryopreservation of their entire structure impossible. Developing effective
cryopreservation protocols for tropical plant species producing recalcitrant seeds is a current
issue and scientific challenge, since during their normal life cycles, they are not exposed to
cold or freezing temperatures or extreme desiccation. Tropical organisms do not, therefore,
possess the natural acclimation responses that can be manipulated to assist cryopreservation,
as is the case for temperate germplasm and cold hardy species (9,21). The fact that many
tropical trees have complex life cycles and only flower and set seed on an infrequent basis,
(sometimes at intervals 5 years) imposes a further conservation problem compounded by
ecological and life cycle factors. Thus, many recalcitrant seeds are irregularly produced in
limited amounts, making their provision as a germplasm source for cryogenic storage very
difficult. Sacrificing seed batches to assess their physiological and storage status compromises
the size of infrequently available seed batches. To overcome this problem, the application of
Taguchi experimental designs for developing cryopreservation strategies for recalcitrant seed
in limited supply has been investigated (19,20). Taguchi-style experiments which employ a
signal to noise ratio as the variable provide similar or more precise conclusions derived from
conducting a fraction of the experiment as compared to full factorial experiments. This
approach may be used for the development of cryopreservation protocols for limited, rare and
at risk germplasm (19,20). Within the same context, the objective of the present study is to
explore alternative strategies that help support the development of cryo-conservation
protocols for storage-recalcitrant genetic resources. In this case, thermal analysis is applied to
help optimise vitrifrication-based cryoprotection strategies with the aim being to integrate
fundamental and applied approaches to assist the development of storage protocols for
tropical trees that produce recalcitrant seeds.
P. speciosa (Leguminosae) is native to Southeast Asia and widely distributed in Southern
Thailand, Peninsular Malaysia, Sumatra, Borneo and the Philippines (26). It produces
recalcitrant seeds and, due to its value and multiplicity of applications as a medicinal and food
plant (15,31) it is important to develop reliable and long-term conservation strategies for its
sustainable utilization. P. speciosa fruit is leathery/woody stalked, comprising an oblong pod
(35-55 cm long and 3-5 cm wide) containing 10-18 (2cm width and 3-5cm diameter) seeds.
These are shed from the mother trees when their moisture content is high (around 80% on a
fresh weight basis). They exhibit typical recalcitrant seed storage behaviour and are extremely
sensitive to desiccation and low temperature (20), making long term storage using
conventional methods impossible.
Due to these problematic issues, a fundamental thermal analysis approach was used to
assist the development of a cryostorage methodology for this species. First, shoot-tips of in
vitro germinated seeds of P. speciosa were chosen for cryopreservation in preference to whole
seeds or excised embryos to advantageously integrate tissue culture manipulations with in
vitro conservation. Furthermore, shoot-tips provide an alternative source of germplasm for
tropical trees producing highly recalcitrant seeds that are difficult to procure, establish and
transfer to seed storage facilities. Secondly, an encapsulation-vitrification (PVS2) method was
selected as this is recommended (22) for highly desiccation sensitive germplasm. Moreover,
this cryoprotective strategy can be used in combination with thermal analysis to study the
effects of several approaches to achieve vitrification. The widespread presence of trehalose in
polar biota and desiccation–tolerant organisms is related to the capacity of this specialised
sugar to support low temperature acclimatisation and tolerance to partial dehydration (17). A
novel component of the study involves the application of trehalose in the cryopreservation of
a tropical plant. As this sugar is produced by stress-tolerant extremophiles, its potential for
simulating acclimation, (as applied in preculture medium and, as an alternative to sucrose) in
tropical species is explored. Differential Scanning Calorimetry (DSC) was utilized to reveal

3. 97
the capability of different and complex cryoprotectant combinations to achieve stable glasses
on cooling and rewarming (2,30). The overall experimental objectives were to elucidate: (i)
multi-component cryoprotective strategies which enhance glass forming capacity and stability
and (ii) their physical thermal attributes critical to survival after exposure to liquid nitrogen
(LN).
MATERIALS AND METHODS
Plant material
Seeds were collected from mother trees near the Kuala Lumpur campus of the Forest
Research Institute of Malaysia. Seeds were extracted from their pods, washed in running
water, surface sterilized (10% v/v sodium hypochlorite solution, Domestos, Lever Bros.) for
10 min and with 0.3% (w/v) boric acid for 1 min followed by rinsing once in 30% (v/v)
ethanol for 1 min and three times in sterile distilled water. Surface sterilized seeds were
cultured on Murashige & Skoog (MS) medium (18) supplemented with 30 g/l sucrose
(Duchefa Biochemie) and 5.5 g/l Plant Agar (Duchefa Biochemie) and incubated in a
temperature-controlled growth room at 26 ± 2ºC with a 12 h (60 µmol.m-2
.s-1
) light/12 h dark
photoperiod.
Cryopreservation treatments
Four weeks after germination, shoot-tips (0.8-1.0 cm) were excised aseptically from in
vitro seedlings and cultured in Petri dishes containing MS medium with three different
concentrations of trehalose (2.5, 5 and 10% [w/v]). The shoot-tips were precultured for one,
two and three days in a temperature-controlled growth room at 26 ± 2ºC with a 12 h (60
µmol.m-2
.s-1
) light/12 h dark photoperiod. The shoot-tips (5 mm) were then excised from
precultured shoots, encapsulated in alginate beads (low viscosity, 3% [w/v] Sigma Chemical)
prepared with 0.4 M sucrose MS medium free of calcium salts) and allowed to polymerise for
20 min in a 0.1 M CaCl2 solution at 26ºC (22). Encapsulated shoot-tips were cryoprotected
with PVS2 (22) [30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) DMSO
(dimethyl sulphoxide) and 0.4 M sucrose] for 30, 60 and 90 min at 0ºC in 1.2 ml Nalgene
plastic cryotubes (five beads / cryotube).
After cryoprotection with PVS2, encapsulated shoot-tips were transferred to cryovials
containing 1 ml of fresh PVS2 solution and plunged in liquid nitrogen (LN). Samples were
then rapidly warmed for 2 min at 40ºC and the bead/shoot-tips rehydrated for 20 min in liquid
MS medium with 1.2 M sucrose. Shoot-tips were recovered by plating on semi-solid MS
medium for 24 h followed by transfer to solid MS medium. Cultures were maintained in the
dark at 18ºC in a growth cabinet for 7 days with change of fresh medium every two days, after
which they were placed in a growth room at 26 ± 2ºC with a 12 h (60 µmol.m-2
.s-1
) light/12 h
dark photoperiod.
Recovery assessment
The affect of each treatment was assessed as the number of shoots surviving on the
recovery medium after eight weeks of culture after retrieval from LN. Recovery was defined
as greening of shoot-tips, leaf expansion and new shoot production.
Thermal analysis
Differential scanning calorimetry incorporating both cooling and warming cycles of
encapsulated PVS2 treated P. speciosa shoot-tips was undertaken using a DSC model Perkin
Elmer DSC with Pyris 7 software. The instrument was calibrated with zinc and indium and
pure water as a standard for cryogenic operations (3). Individual sample beads were placed in

4. 98
aluminium pans, sealed with the aid of a Perkin Elmer crimper and weighed to record fresh
weight values. Scans were performed from +25ºC to –150ºC with a cooling/warming rate of
+ 10ºC min
-1
for control and treated shoot-tips (modified from [3]). The shoot-tips were
precultured for three days on MS medium containing 2.5, 5 or 10% trehalose, encapsulated
and then treated with PVS2 for 30, 60 or 90 min Two shoot-tips (replicates) were used for
each treatment. After thermal analysis, sample pan lids were pierced and the pan together with
the sample dried in an oven at 100°C for a minimum of 24 h to determine sample dry weight.
This allowed the calculation of the total water content of the sample and subsequently the
proportion of frozen and unfrozen water. Thermodynamically, as 1 g of water releases 334.5
joules of heat energy when converted into ice and vice versa (4) the osmotically active water
content of the sample (g osmotically active water per g dry weight) was calculated from the
endothermic–heat changes derived from the melt endotherm during the warming cycle along
with the total water content of the sample. The quantity of osmotically inactive water was
calculated as the difference between total water and osmotically active water contents. The
water content of untreated (control) and treated shoot-tips were measured in the same way.
Data analysis
Data analysis was undertaken using multiple logistic regression (20,25). Twenty shoot-
tips were used for each treatment with three replicates. The primary response was the number
of surviving shoot-tips from the total number of shoot-tips, which follows a binomial
distribution (either survived or not survived). The multiple logistic regression is designed to
identify the relative importance of the factors and interactions (where appropriate) that
significantly reduce (5% significant level for this case) the residual deviance for binomial
response variables. Logistic regression was used in preference to the often-used arcsine
transformation or ANOVA as these assume normality and require a large number of replicates
which are not available for this experiment.
RESULTS
Survival
Survival was assessed as the number of shoots that remained green, producing new leaves
and shoot primordia regrowth (Figure 1). Binary logistic regression analysis showed that
fitting trehalose and its quadratic was not significant (G* = 1.297, df = 2, Model 2, Table 1),
indicating that all trehalose treatments produced the same survival. Above 70% survival was
achieved after trehalose pre-treatment regardless of preculture time (1, 2 and 3 days) before
cryopreservation. Importantly, this confirmed that trehalose at 2.5, 5 and 10% was not
harmful. Similarly, fitting the full quadratic model incorporating trehalose and preculture time
did not have a significant effect on survival before cryopreservation (G* = 4.612, df = 3;
P>0.05, Model 3; Table 1) as compared to Model 2. This indicated that all exposure times (1-
3 days) of preculture and trehalose concentration had similar effects on survival. Adding
PVS2 treatment and its quadratic to the model (Model 4, Table 1), had a highly significant
effect on the model predicting survival (G* = 96.795, df = 2, P<0.001) compared to Model 3,
confirming the expected significant difference in survival before and after PVS2 treatment.
All three levels of PVS2 treatment had different effects on the model predicting survival.
Fitting the full model involving trehalose, preculture time and PVS2 treatment and their
interactions did not significantly effect survival. Survival was high at ca. 90% for the 30 min
PVS2 treatment, but cryoprotectant toxicity was observed as PVS2 exposure was increased
(Table 2). Recovery of 70% of the shoot-tips occurred after a 60 min PVS2 treatment and this
value declined to lethal levels (0-10%) after a 90 min exposure to PVS2.

5. 99
Figure 1. Post-cryopreservation survival as shoot regrowth of P. speciosa at 8 weeks. Scale
bar = 3 mm.
Adding LN to the above model (Model 5, Table 1) significantly improved the Model
predicting survival (G* = 102.169, df = 1, P<0.001) as compared to Model 4, showing that
there was a significant difference in the survival of shoot-tips before and after
cryopreservation. The clear difference in survival before and after cryopreservation was noted
for shoot-tips treated with 30 min PVS2, where survival before LN storage was above 70%
but declined to about 20% after cryopreservation (Table 2).
Table 1. Analysis of deviance for trehalose pre-treatment, PVS2 and liquid nitrogen storage
for P. speciosa shoot-tip survival
Model Deviance
explained
G
D
F
Deviance
difference
G*
DF
difference
P
value
1. Null
2. T + T2
0
1.297
0
2 1.297 2 >0.05
3. T + T2
+ D + D2
+T*D 5.909 5 4.612 3 >0.05
4. T + T2
+ D + D2
+T*D + PVS2 +
PVS22
102.704 7 96.795 2 <0.001
5. T + T2
+ D + D2
+T*D + PVS2 +
PVS22
+ LN
204.873 8 102.169 1 <0.001
T=trehalose; T
2
= quadratic function of T; D=preculture time, D
2
= quadratic function of D; PVS2
2
= quadratic
function of PVS2; LN=liquid nitrogen.

6. 100
Table 2. Survival before (-LN) and after cryopreservation (+LN) (± standard error) of P.
speciosa shoot-tips after various trehalose and PVS2 treatments (n=3)
Treatment Survival (%)
Trehalose (%) Preculture time (day) PVS2 (min) -LN LN
control
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
5
5
5
5
5
5
5
5
5
10
10
10
10
10
10
10
10
10
0
1
1
1
2
2
2
3
3
3
1
1
1
2
2
2
3
3
3
1
1
1
2
2
2
3
3
3
0
30
60
90
30
60
90
30
60
90
30
60
90
30
60
90
30
60
90
30
60
90
30
60
90
30
60
90
96.7 ± 5.8
86.7 ± 5.8
66.7 ± 5.8
0.0 ± 0.0
66.7 ± 5.8
66.7 ± 5.8
6.7 ± 5.8
70.0 ± 10.0
76.7 ± 15.3
6.7 ± 5.8
60.0 ± 10.0
90.0 ± 10.0
0.0 ± 0.0
80.0 ± 10.0
80.0 ± 10.0
10.0 ± 10.0
70.0 ± 0.0
80.0 ± 10.0
10.0 ± 10.0
76.7 ± 5.8
76.7 ± 11.6
0.0 ± 0.0
70.0 ± 10.0
70.0 ± 0.0
6.7 ± 5.8
70.0 ± 10.0
60.0 ± 10.0
0.0 ± 0.0
0.0 ± 0.0
30.7 ±10.0
56.7 ± 5.8
0.0 ± 0.0
6.7 ± 5.8
60.0 ± 10.0
0.0 ± 0.0
13.3 ± 5.8
53.3 ± 15.3
0.0 ± 0.0
26.7 ± 5.8
70.0 ± 10.0
0.0 ± 0.0
20.0 ± 10.0
66.7 ± 5.8
3.4 ± 5.8
26.7 ± 5.8
80.0 ± 10.0
6.7 ± 5.8
23.4 ± 5.8
60.0 ± 10.0
0.0 ± 0.0
20.0 ± 10.0
63.4 ± 5.8
0.0 ± 0.0
23.4 ± 5.8
63.4 ± 5.8
0.0 ± 0.0
Differential Scanning Calorimetry
Thermogram data were interpreted for critical cryopreservation parameters corresponding
to onsets and/or peaks for ice nucleation and melting and glass transitions (Tgs). Some minor
thermal events were noted, particularly on rewarming, which were attributed to putative glass
relaxation events. The measurable parameters included: thermal event temperature, enthalpy
value and the water state composition. Water content was determined as osmotically active
(frozen) and osmotically inactive (unfrozen) water for different treatment combinations. Ice
nucleation onset temperature, midpoint and endpoint data are summarized in Table 3. The
average proportion of osmotically active water as compared to the total water content was ca.
86% for untreated, control shoot-tips, declining to ca. 59% after treatment with 2.5% (w/v)
trehalose and exposure to PVS2 for 30 min (Figure 2, Table 4). Further decline to ca. 48%
and 22% after 5 and 10% (w/v) trehalose preculture, respectively was observed in
combination with a 30 min PVS2 treatment (Figure 2, Table 4). Osmotically inactive water
was detectable following extended exposures (60 and 90 min) to PVS2 treatment, a condition
observed across all trehalose concentrations. Likewise, the average proportion of osmotically
inactive water compared to total water content increased to 41, 52 and 78% after 2.5, 5 and
10% (w/v) trehalose preculture, respectively for the 30 min PVS2 treatment. Following PVS2
exposure for 60 and 90 min the average was ca. 100%.

7. 101
Table 3. Cooling and rewarming thermodynamic properties of alginate-encapsulated shoot-tips of P. speciosa, following trehalose
preculture and PVS2 treatment. Means ± standard errors of the mean are displayed (n=2)
%Trehalose
/ PVS2 (min)
Thermal
cycle
Thermal Event1
Onset
(0
C)
Mid-point (o
C) Endpoint (0
C) Enthalpy
(J.g-1
)
Heat Capacity
(J.g*o
C-1
)
Control
Cooling
Warming
Ice Nucleation*
Ice melt*
-13.80 ± 1.48
1.66 ± 0.13
-16.66 ± 1.49
10.57 ± 0.46
26.16 ± 1.14
16.30 ±0.18
176.51 ± 8.78
150.73 ± 2.60
NA
NA
2.5/30 Cooling
Warming
Tg*
Ice Melt*
-40.22 ± 0.95
-39.56 ± 0.06
-42.79 ± 2.49
-32.54 ± 0.04
-44.75 ± 3.72
-27.31 ± 0.21
NA
61.38 ± 4.53
0.17 ± 0.05
NA
5.0/30 Cooling
Warming
Tg*
Ice Melt*
-50.21 ± 1.07
-35.56 ± 0.26
-54.25 ± 0.93
-21.87 ± 0.24
-57.32 ±0.91
15.03 ± 0.94
NA
24.67 ± 1.04
0.23 ± 0.01
NA
10.0/30 Cooling
Warming Ice Melt*
NA
-38.73 ± 0.45
NA
-29.01 ± 0.18
NA
-26.00 ± 0.11
NA
12.29 ± 0.03
NA
NA
2.5/60 Cooling
Warming Tg*
NA
-39.57 ± 0.43
NA
-33.24 ± 0.23
NA
-29.70 ± 0.31
NA
NA
NA
1.88 ± 0.21
5.0/60 Cooling
Warming Tg#
NA
-40.14
NA
-31.89
NA
-27.72
NA
NA
NA
1.57
10.0/60 Cooling
Warming Tg#
NA
-45.04
NA
-34.78
NA
-30.67
NA
NA
NA
1.06
2.5/90 Cooling
Warming
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
5.0/90 Cooling
Warming
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
10.0/90 Cooling
Warming
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
* = event occurs in both replicates; # = event occurs in one replicate out of two; = stable profile, no event detected in either replicates

8. 102
0%
20%
40%
60%
80%
100%
Water
content
1 2 3 4 5 6 7 8 9 10
Treatment (see Table 4 for details)
Osmotically active water Osmotically inactive water
Figure 2. Summary profiles of osmotically active and inactive water contents calculated as %
of total water content. Details of treatment number codes are shown bold in Table 4 (below).
Table 4. Water composition of encapsulated shoot-tips of P. speciosa following
cryoprotective treatments. Data comprise mean and standard errors (SE), (n=2)
Treatment %Water
Content
(FW)
Total Water
Content
(g H2O [g dwt]
-1
)
Osmotically
Active Water
Content
1
(g H2O [g dwt]
-1
)
Osmotically
Inactive Water
Content
2
(g H2O [g dwt]
-1
)
1. Control 85.9 ± 0.3 6.068 ± 0.154 5.238 ± 0.105 0.830 ± 0.049
2. 2.5% trehalose
+ 30 min PVS2
56.5 ± 1.7 2.305 ± 0.095 1.351 ± 0.083 0.954 ± 0.012
3. 5% trehalose
+ 30 min PVS2
44.2 ± 4.5 1.371 ± 0.862 0.654 ± 0.158 0.717 ± 0.07
4. 10% trehalose
+ 30 min PVS2
26.9 ± 1.9 0.368 ± 0.035 0.081 ± 0.003 0.287 ± 0.032
5. 2.5% trehalose
+ 60 min PVS2
20.5 ± 1.9 0.258 ± 0.030 Not detected 0.258 ± 0.030
6. 5% trehalose
+ 60 min PVS2
17.1 ± 1.3 0.207 ± 0.020 Not detected 0.207 ± 0.020
7. 10% trehalose
+ 60 min PVS2
9.9 ± 0.7 0.110 ± 0.008 Not detected 0.110 ± 0.008
8. 2.5% trehalose
+ 90 min PVS2
6.7 ± 1.5 0.172 ± 0.017 Not detected 0.172 ± 0.017
9. 5% trehalose
+ 90 min PVS2
4.1 ± 0.3 0.094 ± 0.003 Not detected 0.094 ± 0.003
10. 10% trehalose
+ 90 min PVS2
3.7 ± 1.4 0.130 ± 0.015 Not detected 0.130 ± 0.015
• dwt = dry weight

9. 103
Representative thermograms for control shoot tips encapsulated in alginate beads (Figure
3a) demonstrated an ice nucleation peak with a corresponding exothermic enthalpy variation
of 176.51 ± 8.78 J.g-1
(Table 3) during cooling and on melting (Figure 3b) an endothermic
event with enthalpy variation of 150.73 ± 2.60 J.g-1
.
-250
-200
-150
-100
-50
0
50
-150 -125 -100 -75 -50 -25 0 25
Temperature ( o
C)
Heat
Flow
(mW)
Exothermic
Down
0
20
40
60
80
100
120
140
160
180
-150 -125 -100 -75 -50 -25 0 25
Temperature (
o
C)
Heat
Flow
(mW)
Endothermic
Up
Figure 3. DSC cooling (a) and warming (b) thermograms for control (untreated) alginate-
encapsulated P. speciosa shoot-tips. Samples were held at 25ºC for 1 min cooled to –150ºC,
held for 1 min and rewarmed to 25ºC at a rate of ±10ºC per min
Enthalpies of melt endotherms varied proportionally with water content (% fresh weight
basis) for trehalose treatments combined with 30 min PVS2 (Table 3 and Figure 4b). The melt
enthalpy for control shoots was 150.73 ± 2.60 J.g-1
; as bead water content decreased with the
application of 2.5% trehalose, this declined to 61.38 ± 4.53 J.g-1
and thereafter with 5% and
10% trehalose to 24.67 ± 1.04 and 12.29 ± 0.03 J.g-1
, respectively. Ice nucleation was
inhibited when 2.5% trehalose with 30 min PVS2 was applied and a Tg with a midpoint of -
42.795 ± 2.49ºC (Table 3 and Figure 4) was observed; however this Tg was not stable on
rewarming (melting enthalpy 61.38 ± 4.53 J.g-1
).
(a)
(b)
Ice nucleation
Ice melting

10. 104
-20
-10
0
10
20
30
-150 -125 -100 -75 -50 -25 0 25
Temperature (o
C)
Heat
Flow
(mW)
Exothermic
Down
2.5% Trehalose 5% Trehalose 10% Trehalose
0
10
20
30
40
50
60
-150 -125 -100 -75 -50 -25 0 25
Temperature (o
C)
Heat
Flow
(mW)
Endothermic
Up
2.5% Trehalose 5% Trehalose 10% Trehalose
Figure 4. DSC cooling (a) and warming (b) thermograms for alginate-encapsulated P.
speciosa trehalose-treated shoot-tips and cryoprotected with PVS2 for 30 min. Samples
were held at 25ºC for 1 min, cooled to –150ºC, held for 1 min and rewarmed to 25ºC at a rate
of ±10ºC per min.
Similar observations were noted for shoot-tips treated with 5 and 10% trehalose,
presenting warming enthalpies of 24.67 ± 1.04 and 12.29 ± 0.03 J.g-1
, respectively and also
indicating glass instability and the more complex profiles of the warming cycles. Freezing
point depression occurred with 2.5% trehalose at –75ºC and at –85ºC with 5% trehalose
(Figure 4b). A number of minor thermal events were noted in almost all treatments during
both cycles; they may be putative glass relaxation events or localised thermal events
associated with complex interactions between cryoprotectants, alginate and plant tissue.
Increasing PVS2 treatment exposure to 60 min resulted in a more thermodynamically stable
profile without nucleation on cooling. A Tg was observed during rewarming with a midpoint:
-33.24 ± 0.23, -31.89 and -34.78ºC for 2.5, 5 and 10% trehalose, respectively (Figure 5),
although some melting phenomena were observed. Increasing PVS2 treatment time to 90 min
eliminated the active water content (Table 4) consistent with a stable thermal profile during
cooling and subsequent rewarming for all three trehalose treatment concentrations (Figure 6).
(a)
(b)
Glass transitions (Tg)
‘Melts’

11. 105
(b)
-20
-10
0
10
20
30
-150 -125 -100 -75 -50 -25 0 25
Temperature (o
C)
Heat
Flow
(mW)
Exothermic
Down
2.5% Trehalose 5% Trehalose 10% Trehalose
0
20
40
60
-150 -125 -100 -75 -50 -25 0 25
Temperature (oC)
Heat
Flow
(mW)
Endothermic
Up
2.5% Trehalose 5% Trehalose 10% Trehalose
Figure 5. DSC cooling (a) and warming (b) thermograms for alginate-encapsulated P.
speciosa trehalose-treated shoot-tips and cryoprotected with PVS2 for 60 min. Samples
were held at 25ºC for 1 min, cooled to –150ºC, held for 1 min and rewarmed to 25ºC at a rate
of ±10ºC per min.
-20
-10
0
10
20
30
-150 -125 -100 -75 -50 -25 0 25
Temperature (o
C)
Heat
Flow
(mW)
Exothermic
Down
2.5% Trehalose 5% Trehalose 10% Trehalose
0
10
20
30
40
-150 -125 -100 -75 -50 -25 0 25
Temperature (
o
C)
Heat
Flow
(mW)
Endothermic
Up
2.5% Trehalose 5% Trehalose 10% Trehalose
Figure 6. DSC cooling (a) and warming (b) thermograms for alginate-encapsulated P.
speciosa trehalose-treated shoot-tips and cryoprotected with PVS2 for 90 min. Samples
were held at 25ºC for 1 min, cooled to –150ºC, held for 1 min and rewarmed to 25ºC at a rate
of ±10ºC per min.
Stable profiles
(a)
(b)
(b)
(a)

12. 106
DISCUSSION
The objective of this study was to apply thermal analysis to aid the development of
cryopreservation procedures for tropical trees that produce storage recalcitrant seeds. The
potential for using in vitro shoot-tips of P. speciosa as an alternative source of germplasm was
tested as this approach integrates tissue culture and biotechnological improvements with in
vitro cryo-conservation. Moreover, logistically this is an easier option compared to
cryopreserving large and highly metabolically active recalcitrant seeds that are difficult to
procure and maintain viable and quiescent (non-germinating) for the periods of time required
for cryopreservation protocol work up or development. This study demonstrated that shoots of
P. speciosa derived from in vitro-germinated seeds had high survival and shoot proliferation
(>95%).
Survival
Before applying cryogenic treatments, it is first essential to test the effects of
cryoprotective treatments on the survival of shoots and encapsulated shoot-tips. In this study,
this comprised two components: the first tested the effects of trehalose applied as a preculture
additive, the second investigated the effect of PVS2 on the survival of alginate-encapsulated
shoot-tips of P. speciosa excised from the pre-treated shoots.
Low temperature cold hardening and sucrose-simulated cold acclimation are often used to
enable the cryopreservation of shoot-tips from temperate woody perennials (8,9,21). As
tropical species are chill sensitive, this study explored the potential for using trehalose as an
alternative pre-treatment additive, based on its capacity to: (i) enhance cold and desiccation
tolerance in cold-extremophilic organisms (17) and (ii) its propensity for moderating glass
transition temperatures (30). To achieve this aim the effect of trehalose on shoot-tip growth
and development was first tested. P. speciosa shoots were treated with 2.5, 5 and 10% (w/v)
trehalose which was applied in preculture medium for 1-3 days prior to cryopreservation. The
results confirmed that trehalose exposure time and concentrations were not harmful to P.
speciosa since >70% survival was achieved after trehalose pre-treatment, regardless of
preculture time and trehalose concentration before cryopreservation.
Vitrification solutions comprise high concentrations of cryoprotective additives and as
tropical germplasm is particularly sensitive to dehydration, it is important to optimise
exposure to PVS2 to minimise osmotic stress. Survival declined in non-cryopreserved
encapsulated shoot-tips with increasing exposure time of PVS2. When these data were
compared with post-cryopreservation survival data, very low (>20%) survival was observed
after 30 min PVS2 treatment and 60% survival after 60 min PVS2 treatment. There were no
survivors after 90 min PVS2 treatment, regardless of trehalose pre-treatment. These survival
profiles most likely reflect the relationship between two critical point survival factors (1,21)
which are the optimisation of exposure to PVS2 to achieve a stable vitrified state and reducing
the deleterious impact of PVS2 which increases with prolonged exposure to osmotic stress.
These findings concur with previous studies that have used thermal analysis to characterize
the glass forming properties of plant germplasm (7,8,13,28) exposed to various combinations
of vitrification treatments.
It was therefore hypothesized that in the case of P. speciosa shoot-tips: (i) A 30 min
exposure to PVS2 was not sufficient to stabilize the metastable state of vitrified water which
devitrified on rewarming; (ii) Extending PVS2 exposure to 60 min improved post-
cryopreservation survival to ca. 60% and (iii) Extending the treatment to a 90 min exposure
to PVS2, the cryoprotective additives became cytotoxic. Thermal analysis has proved
particularly useful in developing vitrification-based cryostorage protocols for a list of
temperate woody plants (2,9,24). Therefore, the present study proceeded to investigate the

13. 107
role of cryogenic factors as determinants of survival in P. speciosa shoot-tips exposed to
cryoprotective treatments using thermal analysis.
Thermal analysis: trehalose
DSC analysis was done by exposing alginate encapsulated shoot-tips treated with various
cryoprotective strategies to cooling and warming cycles. Clear evidence of ice nucleation
during cooling and ice melting on warming were noted for control shoot-tips. These
thermograms confirmed the existence of non-vitrified water which formed ice during cooling
and melted on rewarming and, as expected this ice was lethal to the shoot-tips.
One of the main objectives of this study was to examine the thermal attributes of
trehalose applied to tropical plant germplasm cryopreservation. This sugar is reported to have
exceptional cryoprotective properties compared to other sugars (e.g. sucrose, fructose and
glucose) and higher Tgs in cooled systems (12,30). Trehalose may thus offer advantages for
the stabilization of dehydration-sensitive, vitrified and metastable tropical plant tissues on
rewarming following exposure to ultra low temperatures (6,32). In this study, more than 70%
survival was achieved after trehalose pre-treatment, regardless of the culture time before
cryopreservation. However, DSC revealed that trehalose did moderate the thermal
characteristics of encapsulated, PVS2-treated shoot-tips of P. speciosa following exposure to
the sugar in the preculture medium. In the case of 30 min PVS2 treatments, the lowest Tg
(-50.21 ± 1.07ºC) was noted for 5% (w/v) trehalose and increased to -40.22 ± 0.95ºC as
trehalose concentration decreased to 2.5% (w/v). Noting that Tg is associated with molecular
mobility (32), it is interesting that the heat capacity of Tgs for shoot-tips treated with 2.5%
and 5% (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 ± 0.05 to 0.23 ±
0.01 J.gºC-1
, respectively. The Tg maximum for the 5% (w/v) trehalose pre-treatment might
indicate water molecules have reduced mobility compared to those shoots treated with 2.5%
(w/v) trehalose, particularly as it has been suggested that there is a strong interaction between
trehalose and water and that the sugar slows down the molecular dynamics of water matrices
(5).
In the present study, the enthalpy of the melt-endotherms varied in proportion to trehalose
concentration, for the 30 min PVS2 treatment. The melt enthalpy for control shoots was >150
J.g-1
and decreased to ca. 60 J.g-1
with 2.5% (w/v) trehalose. For the 5% and 10% (w/v)
trehalose treatments, enthalpy declined to around 24 and 12 J.g-1
respectively. This indicates
trehalose influences the thermal events occurring in P. speciosa shoots exposed to
cryoprotective treatments. Wang and Haymet (30) suggest that trehalose in aqueous solution
reduces the freezing capacity of water as compared to sucrose and this reflects trehalose
solutions having lower latent heats of freezing and melting. Higher melting points and glass
transition temperatures may also influence the stability of vitrified cryopreserved systems.
he bioprotective properties of trehalose may therefore be attributed, in part, to a
reduction in molecular mobility. In support of this, Sakurai et al. (23) consider that all the
hydroxyl groups in trehalose can act as both a proton donor and acceptor. This maximizes
hydrogen bonding with water and may account to some extent for the superior hydration
capabilities and biological properties of the sugar. Fahy et al. (11) also suggested that the
hydrogen-bonding capabilities of cryoprotectants can influence their toxicity and efficacy as
cryoprotectants.
Thermal analysis: alginate-PVS2
Vitrification, as evidenced by a Tg, was achieved after 30 min exposure to PVS2,
regardless of the trehalose concentration in the preculture medium. However, more complex
profiles were obtained in the warming cycle, where an endothermic event was detected,
suggestive of glass destabilization. This event may be due to devitrification and it infers that

14. 108
although on cooling, this system was capable of forming a glassy state, this was highly
metastable. On rewarming, molecular mobility increased in samples in which ice nucleation
and growth was previously arrested and crystallization could be detected in thermograms,
often as minor exothermic events below 0ºC. It is possible that this is a result of ice growth
and/or devitrification, the transition from a glassy to crystalline state. Alternatively this may
also involve the re-crystallization and growth of existing, minute and previously undetectable
ice crystals (27). In the present study, if the 30 min PVS2 treatment was suboptimal, there
may be an increased tendency for minor localised domains within a multi-component system
(e.g. alginate matrix, plants tissue and PVS2) to contain minute ice nuclei or highly unstable
glasses. Thus, if molecular motion increases to a critical point on rewarming and/or if this is
not sufficiently rapid, devitrification and crystallization occur, resulting in lethal intracellular
ice-damage on return to higher sub zero temperatures (16). Dumet et al. (9) reported a glass
destabilization event upon rewarming of 4 h desiccated alginate encapsulated Ribes ciliatum
meristems. However, they noted the absence of glass transitions in the alginate bead and
meristem when these were cooled or rewarmed separately. They postulated that this could be
the consequence of differences between the thermal properties of the alginate bead and the
meristem, which might have promoted the ice nucleation. It was also suggested that a
differential moisture gradient may exist between the tissue and the bead, which could incur
glass destablization on rewarming.
Increased exposure to PVS2 for 60 min achieved a Tg and the glass so formed was stable
on both cooling and rewarming, regardless of the trehalose concentration in the preculture
medium. Longer PVS2 treatments stabilized the vitreous state, presumably by further
reducing molecular mobility, arresting ice nucleation and enhancing overall cell viscosity to a
critical point that the glasses so formed were stable on cooling and rewarming. The 90 min
PVS2 treatment completely removed all active water based on the detection limits of this
system. This was confirmed by DSC thermograms which showed a stable profile with no
thermal events being recorded on either cooling or rewarming. Some very minor endothermic
events were noted in encapsulated shoot-tips exposed to the 60 min PVS2 treatment during
the warming cycle. These could arise as a consequence of devitrification; however, their
enthalpies were very small and, when related to survival data, their deleterious impact was not
evident. Very minor endothermic events were also observed for the for 90 min PVS2
treatment. These may be assigned to glass relaxation phenomena and/or very small changes
in the mobility and composition of different components of the alginate, sugar, cryoprotectant
mixture. It is only the major thermal events that demonstrated a positive correlation between
exposure to the cryoprotective additive PVS2, glass stabilization and survival after cryogenic
treatments. At sub-optimal exposures stable glasses were not formed and at supra-optimal
levels the PVS2 became cytotoxic, presumably because of osmotic stress.
Volk and Walters (29) applied DSC to study the mode of action in PVS2 mixtures and
proposed that its protective properties are multiple and may be assigned to osmotic,
vitrification and colligative factors as well as the fact that the vitrification mixture changes the
freezing behaviour of water. Of particular significance to this present study is their suggestion
the PVS2 solutions restrict the molecular mobility of water molecules, impeding their ability
to nucleate. It thus follows that the application of alginate encapsulation, PVS2 and trehalose
in combination has the synergistic effect of reducing the molecular mobility of water. This
may be used to advantage in recalcitrant and desiccation sensitive tropical plant germplasm
for which achieving minimum levels of dehydration with maximum stabilization of
metastable glasses on rewarming is critical to survival after cryogenic storage.
This study has demonstrated how DSC can be used to elucidate cryogenic behaviour in
combined cryoprotection strategies applied to recalcitrant tropical plant germplasm.
Importantly increased germplasm survival has been systematically associated with a decrease

15. 109
in the enthalpies of ice crystallization/ ice melting events and for vitrification-based protocols
recovery is usually only possible on the production of stable glasses (4,8,10,13,14). In the
future, it may therefore be worthwhile to explore in more detail the application of trehalose
for the stabilization of vitrified recalcitrant plant germplasm.
Acknowledgments: Jayanthi Nadarajan acknowledges financial support from the European
Social Fund for her PhD studies and consumables funding from the EU Quality of Life and
Living Resources project CRYMCEPT, QLK5-CT-2002-01279 in compliance with Work
Packs 1, 8 and 9. The authors acknowledge the kind assistance of Dr Jason Johnston, Mrs
Isobel Pimbley and staff of the Forest Research Institute of Malaysia (FRIM).
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Accepted for publication 5/10/07