The purpose of this research is to explore the extent and significance of possible interacting factors on the viability of stored germplasm. Our work begins with characterizing the kinetics of TAG and water phase changes in peanut (Arachis hypogaea) and Papaya (Carica papaya) seeds equilibrated to different water contents and stored at temperatures between -5 and -80°C. Water and TAG phase was measured using a Perkin Elmer Differential Scanning Calorimeter. Cytoplasm ultra-structure was visualized without chemical fixatives using low temperature scanning electron microscopy (cryo-SEM) performed with a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK)

Ballesteros, Daniel

Molina García, Antonio Diego

Schneider Teixeira, Aline

Walters, Christina

Servicio de Coordinación de Bibliotecas de la Universidad Politécnica de Madrid

ability of ZRA to inhibit ice recrystallization and to reduce ice crystal growth rate
imply on its tremendous potential usages in the ﬁeld of cryopreservation and other
biological and engineering ﬁelds.
Supported by ISF, ERC and IRG.
Conﬂict of interest: None declared.
E-mail address: braslavs@agri.huji.ac.il (I. Braslavsky)
http://dx.doi.org/10.1016/j.cryobiol.2012.07.049
49. Interactions between water and triacylglycerols may explain faster aging rates in
stored germplasm at low temperatures. Aline Schneider Teixeira 1, Daniel Ballesteros 2,
Antonio Diego Molina-Garcia 1, Christina Walters 2, 1 ICTAN (CSIC), Jose Antonio
Novais 10, 28040 Madrid, Spain, 2USDA-ARS-National Center for Genetic Resources
Preservation, Fort Collins, CO 80521, USA
Longevity of orthodox (desiccation tolerant) seeds, pollen and fern spores
characteristically increases with decreasing storage temperature. We have discov-
ered numerous seed and fern spore species that age faster when stored in the free-
zer near 20 C compared to storage at higher or lower temperatures. Seeds
exhibiting this temperature anomaly are considered to be intermediate between
orthodox and recalcitrant physiologies. Intermediate traits are often associated
with high proportions of saturated fatty acids within the triacylglycerols (TAG),
accumulated into cells as food reserves. TAG high in saturated fatty acids crystal-
lize and melt within the temperature range of the observed longevity anomalies
and we have hypothesized a link between TAG phase change and increased dam-
age to germplasm cells. Damage is exacerbated as water content within stored
seeds and fern spores increases from near 5% to about 12%. These observations
have led us to hypothesize that damage results from an interaction between
TAG, water, temperature and time. The purpose of this research is to explore
the extent and signiﬁcance of possible interacting factors on the viability of stored
germplasm. Our work begins with characterizing the kinetics of TAG and water
phase changes in peanut (Arachis hypogaea) and papaya (Carica papaya) seeds
equilibrated to different water contents and stored at temperatures between 5
and  80C. Water and TAG phase was measured using a Perkin Elmer Differential
Scanning Calorimeter. Cytoplasm ultra-structure was visualized without chemical
ﬁxatives using low temperature scanning electron microscopy (cryo-SEM) per-
formed with a Zeiss DSN 960 scanning microscope equipped with a Cryotrans
CT-1500 cold plate (Oxford, UK). The time dependency of phase changes was
assessed over a 3 week period. We report that TAG crystallization in seeds is inde-
pendent of water content and occurs over several days. The amount of water that
freezes in seeds increases with storage duration in a manner consistent with TAG
crystallization kinetics.
Conﬂict of interest: None declared.
Source of funding: This work was funded from project ‘‘CRYODYMINT” (AGL2010-
21989-C02-02), Spanish ‘‘Plan Nacional I + D+i 2008–2011”, M.E.C. and ARS National
Center for Genetic Resources Preservation. A.S. Teixeira was supported by the CSIC,
within the JAE-Pre program, partially funded from the European Social Fund.
E-mail address: teixeiraline@hotmail.com (A.S. Teixeira)
http://dx.doi.org/10.1016/j.cryobiol.2012.07.050
50. Relevance of cell biophysical behaviour and membrane ﬂuidity for explaining freezing
resistance of lactic acid bacteria. Stéphanie Passot 1, Marielle Bouix 1, Julie Gautier 2,
Pascale Lieben 2, Stéphanie Cenard 2, Sarrah Ghorbal 1, Fernanda Fonseca 2, 1 AgroP-
arisTech, UMR 782 Génie et Microbiologie des Procédés Alimentaires, F-78 850 Thi-
verval-Grignon, France, 2 INRA, UMR 782 Génie et Microbiologie des Procédés
Alimentaires, F-78 850 Thiverval-Grignon, France
The cell membrane is the primary site for freezing injury. During cooling, ice
forms outside the cell resulting in an increase of the solutes concentration in the
extracellular medium. This cryo-concentration of solutes induces water transport
from the intracellular medium of cell to the extracellular medium and thus cell
dehydration and cell injury. Cell membrane permeability to water is inﬂuenced
by its ﬂuidity property which is governed by the composition and the structure
of the lipid bilayer. By changing the composition of the medium, the composition
and organisation of the lipid bilayer of the cell membrane will be modiﬁed resulting
in increasing or decreasing freezing resistance. Furthermore, another important
phenomenon occurs during cooling of cell suspensions leading to the modiﬁcation
of membrane ﬂuidity: the lipid phase transition from a liquid crystalline phase to a
gel phase. The liquid crystalline state is characterized by increased lipid head group
spacing, increased disorder in the lipid acyl chains, and a small bilayer thickness. In
the gel phase the lipid head groups are very tightly packed, the lipid acyl chains
become straighter and ordered, and the bilayer thickness increases.
Our objective is to gain a better understanding of the relationships between
membrane ﬂuidity, lipid phase transition, membrane water permeability and freezing
resistance of lactic acid bacteria.
Two suspensions of Lactobacillus bulgaricus CFL1 were produced using either
MRS broth or mild whey based culture media. The freezing resistance of both bac-
terial populations protected with sucrose was evaluated by measuring the cultiva-
bility and acidiﬁcation activity of bacteria before and after freezing at 80 C. The
membrane lipid phase behaviour was studied in situ using Fourier transform infra-
red spectroscopy during freezing and thawing. Membrane ﬂuidity was evaluated
with ﬂuorescence polarization using a lipophilic ﬂuorescent probe embedded in
the membrane, namely the DPH. The ﬂuorescence measurements were performed
on a ﬂow cytometer at various temperatures. Bacterial cell dehydration was inves-
tigated by submitting cells to osmotic stress by increasing the concentration on a
non penetrating solute (sucrose). The changes in cell volume with time were mea-
sured by Coulter techniques and the biophysical parameters of water transport were
estimated.
Bacterial population grown in MRS broth exhibited a better resistance to freez-
ing than bacteria grown in mild whey. This better resistance can be ascribed to a
lower lipid phase transition temperature and higher membrane ﬂuidity. The
detailed analysis of the results is in progress and will enable clariﬁcation of the
relationships between freezing resistance, membrane ﬂuidity and water
permeability.
Conﬂict of interest: None declared.
Source of funding: None declared.
E-mail address: spassot@grignon.inra.fr (S. Passot)
http://dx.doi.org/10.1016/j.cryobiol.2012.07.051
Anthony J. Reardon received the John K. Critser Travel Award
51. A comparative assessment of mitochondria and membrane cryobiological responses of
HUVEC to low temperatures. Anthony J. Reardon 1, Janet A. W. Elliott 2, Locksley E.
McGann 1, 1Departments of Laboratory Medicine and Pathology, University of Alberta,
Edmonton, Alberta, Canada, 2Departments of Laboratory Chemical and Materials
Engineering, University of Alberta, Edmonton, Alberta, Canada
The extent of cellular cryoinjury incurred during cryopreservation protocols is
traditionally assessed with membrane integrity assays, often used as an upper limit
in determining viability. However, exposure of cells to subzero temperatures and
conditions may lead to changes in various aspects of cellular structure and function
that would not be considered in studies that rely on membrane integrity assess-
ments alone. Slower cooling rates in particular have been shown to adversely affect
the metabolism (McGann et al., 1988; and Sherman, 1972) and structural properties
(Tchir et al., 2010) of mitochondria in addition to the occurrence of damage to the
plasma membrane during freeze–thaw stress. The objective of the study was to use
a mitochondrial assay to investigate a secondary aspect of cell response to directly
compare with a traditional assessment of membrane integrity under identical con-
ditions. An interrupted slow cooling method was used to examine the occurrence of
damage from slow cooling to intermediate subzero experimental temperatures, and
damage during rapid cooling when plunged into liquid nitrogen. Human umbilical
vein endothelial cells (HUVECs) were cooled at 0.2 C/min to temperatures between
3 C and 40 C in the absence of cryoprotectant. Samples were then either
thawed directly in a 37 C water bath or plunged into liquid nitrogen before thaw-
ing. The membrane integrity of cells was determined using a combination of the
nucleic ﬂuorescent dyes Syto13 and ethidium bromide, and mitochondrial polariza-
tion was indicated with the cationic carbocyanine dye JC-1. The number of cells
determined to be membrane intact and the number of cells containing polarized
mitochondria decreased with decreasing temperature in directly-thawed samples.
Depolarized mitochondria was found in 50% of cells at 15 C, whereas 50% of cells
were membrane compromised at a lower temperature (30 C). HUVEC plunged
into liquid nitrogen from experimental temperatures showed an increase in the
number of membrane intact cells with decreasing experimental temperature to a
maximum of 40% at 20 C, but <8% of these cells contained polarized mitochon-
dria. Directly thawed samples showed a greater proportion of cells with depolarized
mitochondria at higher subzero temperatures than cells with membrane damage,
indicating that subcellular changes occur in cells under conditions that do not com-
promise the integrity of the plasma membrane. At some experimental tempera-
tures, plunged HUVEC showed signiﬁcant membrane integrity, immediately post-
thaw, but few of these membrane intact cells contained polarized mitochondria.
This demonstrates that damage to organelles and the plasma membrane may occur
Abstracts / Cryobiology 65 (2012) 339–366 355


Interactions between water and triacylglycerols may explain faster aging rates in stored germplasm at low temperatures

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