Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures

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Cryobiology 55 (2007) 1–9

Calorimetric properties of water and triacylglycerols in fernspores relating to storage at cryogenic temperatures q

Daniel Ballesteros a, Christina Walters b,*

a Banco de Germoplasma, Jardı Botanic-ICBiBE, Universitat de Valencia, C/Quart, 80, 46008 Valencia, Spainb USDA-ARS National Center for Genetic Resources Preservation, 1111 So. Mason Street, Fort Collins, CO 80521, USA

Received 16 August 2006; accepted 26 March 2007Available online 14 April 2007

Abstract

Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects oftriacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at �15 �C or liquid nitrogentemperatures. We used differential scanning calorimetry (DSC) to monitor enthalpy and temperature of water and TAG phase transi-tions within spores of five fern species: Pteris vittata, Thelypteris palustris, Dryopteris filix-mas, Polystichum aculeatum, Polystichum

setiferum. The analyses suggested that these fern spores contained between 26% and 39% TAG, and were comprised of mostly oleic(P. vittata) or linoleic acid (other species) depending on species. The water contents at which water melting events were first observableranged from 0.06 (P. vittata) to 0.12 (P. setiferum) g H2O g�1 dry weight, and were highly correlated with water affinity parameters. Inspores containing more than 0.09 (P. vittata) to 0.25 (P. setiferum) g H2O g�1 dry weight, some water partitioned into a near pure waterfraction that melted at about 0 �C. These sharp peaks near 0 �C were associated with lethal freezing treatments. The enthalpy of watermelting transitions was similar in fern spores, pollen and seeds; however, the unfrozen water content was much lower in fern spores com-pared to other forms of germplasm. Though there is a narrow range of water contents appropriate for low temperature storage of fernspores, water content can be precisely manipulated to avoid both desiccation and freezing damage.Published by Elsevier Inc.

Keywords: Differential scanning calorimetry; Ex situ conservation; Fern spores; Germplasm; Relative humidity; Triacylglycerol; Unfrozen water; Watercontent

Ex situ conservation is an important strategy to protectimperiled species of Pteridophytes (ferns). Methodologiesfor long term preservation of fern spores are not well estab-lished and hamper ex situ conservation efforts. Fern sporesdie relatively rapidly under laboratory room conditions[24]. Biochemical and metabolic factors, depletion of respi-ratory substrate, loss of membrane integrity, inactivationof enzymes and growth promoters, and chromosomal aber-

0011-2240/$ - see front matter Published by Elsevier Inc.

doi:10.1016/j.cryobiol.2007.03.006

q This work was funded by institutional sources. Mention of tradenames or commercial products in this publication is solely for the purposeof providing specific information and does not imply recommendation orendorsement by the US Department of Agriculture.

* Corresponding author. Fax: +1 970 221 1427.E-mail address: [email protected] (C. Walters).

rations or other genetic mutations are cited as possiblecauses of the rapid loss of viability during storage ofnon-green spores [5,26]. High respiratory rate or failureto recover photosynthetic activity after desiccation is sug-gested to cause the very rapid loss of viability in greenspores [20,24].

Most of the work on ex situ conservation of Pterido-phytes focuses on determining storage conditions thatcan maintain spore viability for several years. Studiesinclude the effects of storage temperatures routinely usedin germplasm banks (25, 5 or �25 �C) on spores that havebeen dried at ambient room conditions [room temperatureand about 65% relative humidity (RH)], over silica gel (RHranges from 5% to 15%) or placed on nutrient mediumand 1% agar [2,8,9,19,20,23,28,35,37]. The feasibility of

2 D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9

cryopreserving fern spores is demonstrated by reports ofnormal growth after at least six years of storage withinliquid nitrogen [1,3,27,32]. In these reports, the spores werecollected from mature fronds after dehiscence of the spo-rangia (e.g. [19]) and they received no other drying treat-ment. Cryostorage, therefore, offers a promising methodto preserve valuable germplasm of ferns, once methods tooptimize water status have been developed.

Establishment of routine cryostorage methodologies forfern spores requires an understanding of the interrelation-ships between water content, temperature and viabilitywithin diverse species. Spores must be dried sufficiently toavoid freezing damage when exposed to subzero tempera-tures, but damage by desiccation must also be avoided. Tol-erance to drying over silica gel appears to vary amongspecies, with both survival [3,9,35] and damage [22,23,28]reported. Fern spores collected at room conditions aresometimes killed by exposure to �25 �C [2,3,28,35], anddrying over silica gel before placing spores in the freezerincreases survival [3,9]. Occasional damage when sporesare dried or cooled to extreme levels have prompted theuse of alternative storage methods, such as hydrated storageon nutrient medium [2,19,23,28]. We propose that damageto spores by desiccation or cooling can be prevented by pre-cise control of the water content within spores. In a previousstudy [4], we showed that there is a very narrow window ofwater contents appropriate for storage of fern spores at con-ventional storage temperatures. In this study, we considerthe added effects of cryogenic temperatures on water andlipid phase changes. The combination of the two studies willallow us to predict water content and temperature combina-tions that avoid desiccation and freezing damage and pro-mote long term viability of fern spores.

The purpose of this paper is to determine the interac-tions of water content and temperature on phase transi-tions that may be important to cryostorage of fernspores. We focus on water and triacylglycerol (TAG) crys-tallization events, which are readily detected using differen-tial scanning calorimetry (DSC). It is generally acceptedthat intracellular ice formation is lethal, and that the watercontent of cells and cooling rate to liquid nitrogen temper-atures can be balanced to avoid dangerous ice crystals[41,51–54]. Recent studies have also linked TAG crystalli-zation to damage in seeds exposed to conventional freezerstorage and have suggested that this is the basis for so-called intermediate storage physiology [10,11,45]. Seedswith intermediate storage behavior do not survive the com-bined effects of low moisture and low storage temperatures[13], and some of the empirical data on fern spore longevitysuggest a similar category exists in ferns [2,3,22,23,28,35].

Materials and methods

Plant materials and water content determinations

Mature fronds of different fern species were collectedfrom wild populations during the summer in 2005 at the

Valencian Community, Spain (Table 1). Fronds werepressed onto glossy paper and allowed to dry (e.g. [19]).Spores were collected from the sheets after sporangialdehiscence, sieved and subsequently stored at �80 �C untilused. Spores were mailed to Fort Collins, CO, USA usingexpedited post and arrived within 3 days.

Water content of spores was manipulated by placingthem in RH chambers at 25 �C or over water vapor. RHwas adjusted using saturated salt solutions [31,44,55]. Afterwater contents were adjusted, about 1–10 mg of fern sporeswere sealed into aluminum DSC pans. Standard 20 ll pansthat seal hermetically were used for spores adjusted tohigher water contents. Spores containing less than0.08 g H2O g�1 dry weight (RH < 85%) were packed intonon-hermetically sealing 40 ll pans to improve thermalconductivity within the DSC. After DSC analysis, panswere punctured and placed at 95 �C for 36 h, a time suffi-cient to achieve constant weight. Water contents were cal-culated from the difference in fresh and dry weights and areexpressed on a dry weight basis as g H2O g�1 dry weight(dw).

Differential Scanning Calorimetry

Phase transitions in fern spores at various water con-tents were determined using a Perkin–Elmer (Norwalk,CT) DSC-7, calibrated for temperature with methylenechloride (�95 �C) and indium (156.6 �C) standards andfor energy with indium (28.54 J g�1).

The presence of phase transitions was determined fromcooling and heating thermograms recorded between �150and +40 �C while scanning at a rate of 10 �C min�1. Theonset temperature of the melting and freezing transitionswas determined from the intersection between the baselineand a line drawn from the steepest portion of the transitionpeak. The enthalpy (DH) of the transition was determinedfrom the area encompassed by the peak and the baseline.All analyses were performed using Perkin-Elmer software.Enthalpies of exothermic and endothermic events areexpressed on a per g dry weight basis. The temperatureand DH of transitions at very low water contents did notchange with slight increases in water content and so wereattributed to triacylglcerols (TAG) [11,49]. Triacylglycerolswith b 0 melting transitions in the temperature rangesobserved for fern spores are triolein (�12 �C), trilinolein(�23 �C) and trilinolenin (�34 �C) [36]. As water contentsprogressively increased, transition size increased, and theslope of the linear relationship was used to calculate theDH of the water melting or freezing transition on a g�l

H2O basis (e.g., [40,46]). The intersection of this line withthe horizontal one attributed to TAG transitions revealedthe water content below which melting or freezing transi-tions could not be observed [40]. At higher water contents,water melting transitions were partitioned into a broadpeak with onset less than �10 �C and into a sharp peakwith onset close to 0 �C. The DH of the sharp peak was cal-culated separately and regressed against water content to

Table 1Collected species

Species Population Order and Family Ecology Spore type and Ploidy

Dryopteris

filix-mas

Maset del Zurdo Vistabella del Maestrat, Castellon.Spain. 1380 m

Aspidiales andAspidiaceae

Forest understoryshade

Monolete andpolyploid 2n = 164

Polystichum

aculeatum

Font del Tilde Maset del Zurdo Vistabella delMaestrat, Castellon. Spain. 1200 m

Aspidiales andAspidiaceae

Forest understoryshade, moist

Monolete andpolyploid 2n = 164

Polystichum

setiferum

Barranco del Juncaret Ahin, Castellon. Spain. 650 m Aspidiales andAspidiaceae

Forest understoryshade, moist

Monolete and diploid2n = 82

Pteris vittata Barranco de la Safor Villalonga, Valencia. Spain.260 m

PteridalesandPteridaceae

Tropical moist Trilete and polyploidy

Thelypteris

palustris

Ullals Riu Verd Massalaves, Valencia. Spain. 35 m Aspidiales andThelypteridaceae

Moist Monolete and Diploid2n = 70

D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9 3

calculate melting DH on a g�l H2O basis (slope) and theminimum water content at which these sharp peaks weredetectable (x intercept).

Results

The physical state of water and TAG in homosporeous,non-green spores of five fern species was studied using dif-ferential scanning calorimetry. We measured the tempera-ture and enthalpy of phase transitions that occurred

D. filix-mas

T. palustris

P.vittata

P.aculeatum

P.setiferum

0-75-100-125-150 -25-50

0

2.0

1.0

b

Temperature (oC)

Po

wer

(m

Wg

-1d

w)

En

do

ther

mic

D. filix-mas

P. vittata

T. palustris

P. aculeatum

P. setiferum

0

0.4

0.8

1.2 a

Fig. 1. DSC cooling (a) and heating (b) thermograms of spores fromdifferent fern species equilibrated to water contents between 0.02 and0.035 g water g�1 dry weight. Samples were scanned at 10 �C min�1 from40 to �150 �C.

between �90 and 10 �C as a function of the water contentof the spores.

Phase changes were detected when dry spores werecooled to �150 �C (Fig. 1). DSC scans in Fig. 1 are forspores containing between 0.02 and 0.035 g H2O g�1 dw,depending on species; however, similar scans were obtainedfor spores that were dried to less than 0.005 g H2O g�1 dwand for spores containing about 0.08 g H2O g�1 dw. Exo-thermic events were detected during cooling runs between�20 and �90 �C (Fig. 1a) and corresponding endothermicevents were observed during warming between �35 and+10 �C (Fig. 1b). Several species demonstrated multiplecrystallization and melting transitions during cooling andheating, respectively. Onset temperatures for melting tran-sitions (endothermic events) in dry spores were highest forP. vittata (��12 �C) and lowest for T. palustris (��34 �C).The average enthalpy of the melting transitions was about30 J g�1 dw and ranged from 23 J g�1 dw for T. palustris to34 J g�1 dw for D. filix-mas (Table 2). The size, tempera-ture and lack of response to water content suggest thatthese transitions arise from crystallization and melting ofTAG [10,11,39,49]. The melting temperature of lipids inP. vittata is comparable to the melting temperature of b 0

crystals of triolean (�12 �C according to [36]), suggestingthat spores of this species contain TAG with high oleic acidcontent. Assuming complete crystallization of TAG and amelting enthalpy of 100 J g�1 lipid [36], we can estimatethat about 33% of the dry weight of spores of this speciesis lipid (Table 2). Lipids of the other species can be pre-dicted to contain more linoleic or linolenic acids based onthe melting temperatures. Lipid contents for these speciescan be estimated, based on melting enthalpies for simpleTAG [36], to range from 26 (T. palustris) to 39%(D. filix-mas) (Table 2).

As water contents were progressively increased, otherexothermic and endothermic events were evident in DSCscans (Figs. 2 and 3). Initially, the area encompassed bythe lipid transitions increased preferentially (e.g., comparecooling and warming scans of D. filix-mas spores contain-ing 0.12 g H2O g�1 dw in Fig. 2 and for T. palustris sporescontaining 0.18 g H2O g�1 dw in Fig. 3). As spores became

Table 2A summary of the calorimetric properties measured for lipid and water in fern spores

Component Parameter P. aculeatum P. setiferum D. filix-mas T. palustris P. vittata

Lipid Melting temperature (�C) �26, �4.1 �24.8 �26.3 �33.6, �11.3 �11.8, �6.7Melting enthalpy (J g�1 dw) 31.9 ± 1.7 27.7 ± 0.4 34.2 ± 0.9 22.9 ± 3.9 32.5 ± 0.9Predominant fatty acid (predicted) Linoleic Linoleic Linoleic Linolenic OleicEnthalpy of melt J g�1 lipida 88 88 88 88 100Lipid content % (estimated) 36 32 39 26 33

Water Unfrozen water (g H2O g�1 dw) 0.084 + 0.018 0.121 + 0.021 0.088 + 0.018 0.082 + 0.018 0.060 + 0.011Melting enthalpy (J g�1 H2O) 214 + 11 234 + 11 244 + 16 245 + 14 233 + 16Freezing transition (�C) �18 �18 �14.5 �23 �25Limit of sharp peak (g H2O g�1 dw) 0.136 + .043 0.252 + 0.092 0.119 + 0.068 0.182 + 0.056 0.092 + 0.055Sharp peak enthalpy (J g�1 H2O) 78 + 16 98 + 19 54 + 12 53 + 9 68 + 23

BET model at 25 or5 �Cb

Monolayer value at 25 �C(g H2O g�1 dw)

0.027 0.034 0.026 0.028 0.022

Monolayer value at 5 �C(g H2O g�1 dw)

0.035 0.040 0.032 0.037 0.027

BETc at 25 �C (c � exp(DHsorp/RT)) 80 54 63 81 79BETc at 5 �C (c � exp(DHsorp/RT)) 308 401 110 77 100

a [36].b [4].

b

0-75-100-125-150 -25-500

2.0

1.0

3.0

4.0

a

0.29 g g-1

0.12 g g-1

0.07 g g-1

0.02 g g-1

0.19 g g-1

2.0

1.0

3.0

4.0

0

En

do

ther

mic

L L

w sp

sp

sp

w

L

0.29 g g-1

0.12 g g-1

0.07 g g-1

0.02 g g-1

0.19 g g-1

Temperature (oC)

Po

wer

(m

Wg

-1d

w)

Fig. 2. DSC cooling (a) and heating (b) thermograms of Dryopteris filix-mas

spores containing indicated amounts of water. Samples were scanned at10 �C min�1 from 40 to�150 �C. Transitions are identified as lipid (L), water(w), and water with melting temperature corresponding to pure water (sp).

b

0-75-100-125-150 -25-500

2.0

1.0

3.0

4.0

a

2.0

1.0

3.0

4.0

0

En

do

ther

mic

0.37 g g-1

0.11 g g-1

0.07 g g-1

0.26 g g-1

0.18 g g-1

0.11 g g-1

0.03 g g-1

LL

wsp

LL

w

sp

0.37 g g-1

0.07 g g-1

0.26 g g-1

0.18 g g-1

0.11 g g-1

0.03 g g-1

0.11 g g-1

Temperature (oC)

Po

wer

(m

Wg

-1d

w)

Fig. 3. DSC cooling (a) and heating (b) thermograms of Thelypteris

palustris spores at different moisture contents. Samples were scanned at10 �C min�1 from 40 to �150 �C. Transitions are identified as lipid (L),water (w), and water with melting temperature corresponding to purewater (sp). Thermogram characteristics presented for this species or forD. filix-mas (Fig. 2) are representative of thermograms acquired for otherspecies.

4 D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9

increasingly hydrated, sharp exothermic peaks wereobserved during cooling, and broad peaks with onset atabout �10 �C and sharp peaks at about 0 �C were evidentupon warming (Figs. 2 and 3). The size of the peaks and theonset temperature increased as the water content within thespores increased. These exothermic and endothermic eventswere attributed to ice formation and melting [40].

The effect of water content on crystallization and melt-ing enthalpy can be visualized in fern spores from plotsof enthalpy, calculated on a g�1 dry weight basis, versus

D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9 5

water content, expressed on a g�1 dry weight basis (meltingtransitions given in Fig. 4). At low water contents, whenonly the lipid transitions were detected, DHm was constantwith water content. Once spores were sufficiently hydrated,DHm increased linearly with increasing water content(r2 > 0.90 for regressions from all species). The values forDHm (slope of linear regression) ranged from214 J g�1 H2O for spores of P. aculeatum to 245 J g�1 H2Ofor spores of T. palustris (Table 2). The amount of waterthat did not freeze was calculated from the intersection ofa horizontal line drawn for lipid transitions and the slopedline drawn for water transitions. Unfrozen water contentsaveraged 0.087 g H2O g�1 dw across the species studiedand ranged from 0.060 g H2O g�1 dw for spores of P.vitat-ta to 0.121 g H2O g�1 dw for spores of P. setiferum (Table2).

The enthalpy associated with the sharp melting peak at0 �C was also expressed as a function of spore water con-tent (Fig. 4; r2 > 0.85 for all regressions except for P. vittata

where r2 = 0.68). The slopes of this relationship averaged72 J g�1 H2O and ranged from 98 to 53 J g�1 H2O(Table 1). Sharp melting peaks at 0 �C were usually notobserved in spores containing less than 0.12 g H2O g�1 dryweight, but the limits of detection ranged from0.09 g H2O g�1 dw for spores of P.vitatta to 0.25 g H2Og�1 dw for P. setiferum.

A summary of calorimetric data is presented in state dia-grams where temperatures for lipid and water crystalliza-tion events are expressed as a function of fern sporewater content (Figs. 5–7). In cases where transition onsets

T. palustris

53 J g-1 H2O

sharpeak

watermelt

Water conte

Tra

nsi

tio

n e

nth

alp

y (J

g-1

dw

)

0 0.1 0.2 0.3

80

40

100

60

20

0

80

40

100

60

20

0

D. filix-mas

245 J g-1 H2O

54 J g-1 H2O

sharppeak

watemelt

244 J g-1 H2O

Fig. 4. The relationship between the water content and enthalpy of melting tcalculated from the sum of all endothermic peak areas observed in thermograms(diamonds) was calculated from separate area calculations of the endothermicpoints at higher water contents or the average enthalpy at the lower water contethe melting transition on a per g water basis. Arrows indicate the minimum wvalue is said to be unfrozen. Data for P. aculeatum spores are not shown but

were masked by concurrent events, dotted curves describethe presumed relationship. For example, low enthalpywater melting events in D. filix-mas spores containing0.09–0.18 g H2O g�1 dw were masked by the lipid meltingevent that occurred between �26 and �5 �C (Figs. 2band Fig. 5). In fully hydrated spores, onset of water crystal-lization occurred near �16 �C for spores of P. aculeatum,

P. setiferum and D. filix-mas and below �23 �C for sporesof T. palustris and P. vittata. Onset of water melting eventsin fully hydrated spores was usually between �8 and�4 �C. Partially drying spores caused a decrease in onsettemperatures, as expected, for all species except P. vittata.In this species, onset of water melting transitions wasmasked by the lipid melting transitions that occurred attemperatures >�12 �C. The temperature of the sharp waterpeak in warming scans was constant at 0 �C for all speciesthroughout the entire moisture range at which it wasdetected. The state diagrams also show that transition tem-peratures for TAG are fairly independent of temperature,except for P. vittata where a slight decrease in crystalliza-tion temperature was noted with increasing water content(Fig. 7). Finally, an isopleth (water content-temperaturecombination) corresponding to RH = 20% is included onthe state diagrams as a reference to the presumed optimumwater content for storage according to [47] and [4].

Discussion

We have characterized phase behavior of water andTAG in fern spores from five species. This information

p

nt (g H2O g-1 dw)

0 0.1 0.2 0.3

233 J g-1 H2O

63 J g-1 H2Osharppeak

watermelt

P. vittata

P. setiferum

234 J g-1 H2O

98 J g-1 H2O

watermelt

sharppeak

r

ransitions for different fern spores. Total melting enthalpy (squares) wassimilar to those given in Fig. 1. Enthalpy of the sharp melting peak at 0 �C

peak occurring near 0 �C. Lines represent the least-squares best fit of datant range. The slopes of the lines are indicated and represent the enthalpy ofater content for detecting water melting transitions and water below thatare similar to data presented for P. setiferum.

Water content (g H2O g-1 dw)

0 0.10 0.20 0.30

0

20

-20

-40

-60

Tem

per

atu

re (

oC

)

D. filix-mas

0

20

-20

-40

-60

P. aculeatum

Fig. 5. Phase diagrams for spores of P. aculeatum and D. filix-mas,describing the interaction between water content and temperature on lipidcrystallization and melting temperatures (triangles), water transitiontemperatures (circles), and water transitions that occur within a narrowtemperature range around 0 �C (squares). Open symbols representcrystallization events and solid symbols represent melting events. Datawere determined from thermograms similar to those in Figs. 1–3. Thedashed curve, calculated from isotherms [4], represents the water content-temperature isopleth at 20% RH, which is hypothesized to be the limit ofdesiccation tolerance. Dotted lines are presumed relationships betweentemperature and water content that are masked in thermograms becauseof other concurrent events.

0 0.10 0.20 0.30

0

20

-20

-40

-60

T. palustris

0

20

-20

-40

-60

P. setiferum

0.40

Water content (g H2O g-1 dw)

Tem

per

atu

re (

oC

)

Fig. 6. Phase diagrams for spores of P. setiferum and T. palustris. Symbolsare described in the legend for Fig. 5.

0

20

-20

-40

-60

0 0.05 0.10 0.15

P. vittata

Water content (g H2O g-1 dw)

Tem

per

atu

re (

oC

)

Fig. 7. Phase diagrams for spores of P. vittata. Symbols are described inthe legend for Fig. 5.

6 D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9

can be used to optimize moisture treatments of fern sporesbefore they are exposed to cryogenic storage as well as topredict longevity behavior.

Lipid transitions were detected in DSC scans and weestimated that 25–40% of the dry mass of spores wasTAG based on the enthalpy of the melting signals (Table2). This estimate is consistent with earlier reports thatTAG comprise between 20% and 50% of the dry mass ofnongreen spores from a variety of fern species[12,15,16,25,29,30,34,38]. Transition temperature is relatedto fatty acid chain length and degree of saturation[10,11,36,45]. Comparisons among the species tested sug-gested that T. palustris spores have the highest amount ofpolyunsaturated fatty acids and P. vittata spores have thehighest amount of monounsaturated fatty acids (Table 2).Again, this is consistent with previous reports that oleicand linoleic acids are the predominant fatty acids extractedfrom fern spore TAG [15,30]. Degree to which fatty acidsare unsaturated has been correlated with poor shelf life[17], although the same trends do not appear to hold inseeds (Walters, unpublished observation).

Water melting transitions were detected in fern sporescontaining more than about 0.09 g H2O g�1 dw (Table 2).Thus, the unfrozen water content, when corrected for thepresumed lipid content, was between 0.09 and0.18 g H2O g�1 nonlipid dry matter for spores of P. vittata

and P. setiferum, respectively, and corresponded to watercontents achieved by equilibrating spores to 89–91% RHat 25 �C [4]. Unfrozen water contents range between 0.25and 0.30 g H2O g�1 nonlipid dry matter for most plant

D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9 7

germplasm [6,18,21,33,40,41,50,51,56], except for thosewith renowned poor storage behavior, namely Typha latifo-

lia pollen, and mature embryos of Zizania, Aesculus andCitrus spp [7,14,18,21,42]. The RH corresponding to unfro-zen water contents is about 90% RH at 25 �C for mostgermplasm, and between 75–80% at 25 �C RH for pollenand embryos of Zizania, Aesculus and Citrus spp.[6,7,14,18,20,21,33,40–42,50–54]. Thus, the low unfrozenwater contents observed in fern spores is primarily a resultof the low water contents achieved when equilibratingspores to 90% RH.

The low amount of water absorbed in fern spores com-pared to other germplasm equilibrated to similar RH ledus to propose that fern spores had a low affinity for water[4]. Isotherm parameters, such as the Brunauer–Emmett–Teller (BET) monolayer value describe water affinity[31,47,57]. BET monolayer values were low for fernspores compared to pollen and seeds and ranged amongspecies of ferns. Spores of P. setiferum had the highestBET monolayer value (=0.034 g H2O g�1 dw) and sporesof P. vittata had the lowest (=0.022 g H2O g�1 dw) [4].Differences in unfrozen water content among species fol-lowed a similar trend and was highly correlated with theBET monolayer (r2 = 0.93 for BET monolayer valuesmeasured at 25 �C).

The sharp water melting peak at 0 �C has been attrib-uted to departitioning of water through a eutectic or otherphysical process. It is particularly evident in immatureembryos that are highly vacuolated [6,14,42] and its occur-rence was linked to slow cooling and lethal freezing injury[41,51]. The difference between water contents at whichmelting transitions are detected and sharp peaks at 0 �Care detected provides some insights on relative coolingrates necessary when exposing germplasm to cryogenictemperatures. The appearance of the 0 �C sharp peaks atwater contents only 0.03–0.04 g H2O g�1 dw greater thanthe unfrozen water content in spores of D. filix-mas, P. vit-

tata and P. aculetaum (Table 2) suggests that successfulcryoprotective treatments to spores of this species will nec-essarily involve drying to water contents less than theunfrozen water content, cooling much faster than the10 �C min�1 used in DSC measurements, or exposingspores to exogenous cryoprotective solutions [46].

The enthalpy of melting transitions may also revealproperties of water and ice relevant for cryopreservation.Pure water melts with an enthalpy of 333 J g�1 H2O, anda slight reduction of this value is expected as melting tem-perature decreases [40]. Lower enthalpies may reflect thepresence of solutes or matrices that limit ice formation orpromote recrystallization. For example, water meltingenthalpies of less than 50 J g�1 H2O were measured in mer-istems cryoprotected using plant vitrification solution 2(PVS2) [46]. Germplasm with naturally acquired tolerancesto low temperature or desiccation exhibits melting enthal-pies of 150 ± 50 J g�1 H2O at water contents near theunfrozen water content, but this value increases to near300 J g�1 H2O as water content increases [7,14,40,42]. In

immature or highly recalcitrant embryonic axes, whichhave low tolerance to stresses, melting enthalpy is near300 J g�1 H2O at water contents above the unfrozen watercontent [6,14,41,42,51]. Melting enthalpy in fern spores(�230 J g�1 H2O) is lower than expected for pure water,but higher than the �150 J g�1 H2O commonly observedin other relatively dry germplasm (Table 2). The intermedi-ate values for melting enthalpy suggest limited endogenousprotection from ice crystallization and growth during cryo-exposure of fern spores.

Residual water in dry cells does not freeze when exposedto freezing temperatures because the water molecules aresufficiently immobilized to preclude the molecular reorga-nization required for a crystallization event [56]. That is,water molecules are restricted by the formation of a glass.Restricted mobility within a glass has recently been corre-lated with the enthalpy of sorption, calculated from thenatural logarithm of the parameter c in BET analyses ofisotherms [57]. We find a high correlation between c calcu-lated from 5 �C isotherms and the enthalpy associated withwater melting in the sharp peak at 0 �C (r2 = 0.92) (seeTable 2 for values). Unfrozen water content, calculatedon a lipid free basis, correlated moderately with c calcu-lated from 25 �C isotherms (r2 = 0.77). Future research willinvestigate the links between glass behavior detected usingisotherm and calorimetric data.

Collectively, the calorimetric properties and isothermdata in fern spores describe narrow allowances for watercontent during effective storage. Based on the phase dia-grams given in Figs. 5–7, it is not surprising that freezerstorage (�15 to �25 �C) of spores has provided mixedresults [2,3,9,28,35]. To avoid freezing damage, fern sporesplaced in the freezer must be stored at water contents lessthan the unfrozen water content (about0.09 g H2O g�1 dw). To avoid desiccation damage, fernspore water content should be above a critical level.Research is underway to establish the value of the criticalwater content, and we have hypothesized that it corre-sponds to the BET monolayer value or a water content cor-responding to 20% RH [4] as shown for seeds and pollen[7,43,47,48]. The water content corresponding to a specificRH or the BET monolayer increases with decreasing tem-perature [4], resulting in an ever narrower window of allow-able water contents as storage temperature decreases (seeFigs. 5–7). The upper limits of this window can be expandedby rapid cooling techniques and storage at liquid nitrogentemperatures [51–54]. The tendency for TAG crystallizationduring freezer storage may be a further complication thatneeds consideration when developing routine preservationprotocols for fern spores.

Water contents of fern spores prepared for freezer orcryogenic storage can be fine-tuned using water sorptionisotherms [4]. Depending on species, the window of allow-able water contents for storage at �15 to �25 �C is pre-sumed to occur between 0.04 and 0.09 g H2O g�1 dryweight and can be achieved by equilibrating fern sporesto RH between 50% and 70% at 25 �C.

8 D. Ballesteros, C. Walters / Cryobiology 55 (2007) 1–9

Conclusions

Assessments of phase changes of water and neutral lip-ids within fern spores hydrated to different water contentsand cooled to �150 �C allow us to critically examine thefeasibility of maintaining viability using different storagestrategies. We show that there is a narrow range of watercontents that prevents freezing injury while minimizing des-iccation damage, and that these water content ranges varyamong species. Despite these limitations, optimized watercontents can be achieved for diverse species by equilibrat-ing spores at room temperature to a specific RH. Thismeans that routine preservation protocols to accommodatediverse species can be established. The stringency of precisecontrol of water content can be somewhat ameliorated bycryogenic storage.

Acknowledgments

D.B. was supported by a FPU Grant from the SpanishMinistry of Science and Technology, project REN 2002-03697 (directed by A.M. Ibars). Collections were madeby the Germplasm Bank of the JBUV (directed by E. Estr-elles). The authors are grateful for the support of Drs.Ibars and Estrelles and to Lisa Hill for her expert technicalassistance.

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