Abstracts / Cryobiology 61 (2010) 362–408
the storage outcome (Harding et al., 2009, Agriculture and Food Science 18, 3–16). Of
importance are: selection of donor material (genotype, physiological condition); tis-
sue type (explant), pregrowth conditions, pretreatment, cryoprotection, cooling
(rapid, slow), rewarming, thawing, recovery, plantlet regeneration, acclimation and
ultimately ex vitro establishment and continued growth and development to repro-
ductive maturity. Therefore, cryogenic and non-cryogenic factors influence the
long-term recovery of clonally propagated crops and understanding their impacts
on maturation and reproduction is critical for the successful reintroduction of plants
recovered from cryobanks in breeding programmes (Harding and Staines, 2001, Cryo-
Letters 22, 255–262).
This study considers a number of variables that potentially influence the cryostor-
age of Allium sativum (garlic). The impact of time and temperature on the post-harvest
storage period and the effect of breaking bulb dormancy were evaluated. The rele-
vance of these factors in establishing: (1) an effective micropropagation regime and
(2) the significance of these findings for cryopreservation by the encapsulation/dehy-
dration of A. sativum in vitro cultures will be presented.
It is crucial to consider the effects of the adaptive physiological changes that
occur during the seasonal cycle of Allium species and their consequences for recovery
following cryostorage. This is especially important, as the time of year when Allium
material is harvested; along with the storage regime and duration of storage have
dramatic effects on the response of tissues to in vitro culture. The physiological (epi-
genetic) state of explants taken from adult plants at different times of the year vary
considerably according to the genotype and perceived environmental signals which
determine proliferation in vitro. Morphogenetic changes of Allium bulbs including
the physiological mechanisms of dormancy and sprouting are important post-harvest
storage factors. The dormancy period is highly variable, in which the temporary delay
of meristematic growth is an integral part of the annual life cycle in Allium. In view of
these factors, it is important that future studies consider the potential impact that cli-
mate change may have on the cryostorage of clonally propagated temperate crops
(Lynch et al., 2007, J. Hort. Science & Biotech. 82, 157–160).
Funding: EU COST Action 871 CRYOPLANET; EU CRYMCEPT QLK5CT-2002-01279.
Acknowledgements: Dr J. Keller, IPK, Germany, for garlic germplasm; Keith Har-
ding for a Visiting Research Fellowship at the University of Derby.
Conflict of interest: None declared.
Source of funding: None declared.
doi:10.1016/j.cryobiol.2010.10.131
128. Effect of osmotic pretreatments on oxidative stress and antioxidant profiles in olive
somatic embryos cryopreserved using controlled rate cooling. Paul T. Lynch 1, Ayesha
Siddika 1, Jason W. Johnston 2, Susan M. Trigwell 3, Aradhana Mehra 1, Carla Benelli 4,
Maurizio Lambardi 4, Erica E. Benson * 1,5, 1 Biological Sciences Research Group, Uni-
versity of Derby, Kedleston Road, Derby DE22 1GB, UK, 2 The New Zealand Institute
for Plant and Food Research Ltd., 120 Mt Albert Rd, Private Bag 92 169, Mt Albert,
Auckland, New Zealand, 3 FRAME, Russell and Burch House, 96-98 North Sherwood
Street, Nottingham NG1 4EE, UK, 4 Istituto per la Valorizzazione del Legno e delle
Specie Arboree, CNR, via Madonna del Piano 10, 50019 Sesto Fiorentino (Firenze),
Italy, 5 Damar Research Scientists, Damar, Drum Road, Cupar Muir, Fife, KY15 5RJ
Scotland, UK
Cryopreservation provides a complementary approach to conserving olive
(Olea europaea L.) genetic resources as clonal field collections (Fabbri et al., 2009.
in: Jain, M., Priyadarshan, M. (Eds.), Breeding Tropical Tree Crops. Springer, Berlin,
pp. 423–465). This study combines osmotic pretreatments with controlled rate cool-
ing, as an alternative method to cryopreserving olive germplasm using vitrification or
encapsulation. The objectives are to enhance the efficiency of olive cryobanking for
large-scale culture collections by: (1) optimizing preculture treatments and (2)
improving recovery after controlled rate cooling and cryostorage.
A three-day pretreatment of olive somatic embryos (SE) with 0.75M sucrose, fol-
lowed bycryoprotection with 0.5M DMSO, 1M sucrose, 0.5M glycerol and 0.009M pro-
line and controlled rate cooling ( 0.5 °C min 1 to 35 °C; incorporating a hold at
35 °C for 35 min) supported regrowth and SE development after cryopreservation.
Profiles of sugars, proline, antioxidant enzymes, Reactive Oxygen Species (ROS), sec-
ondary oxidation products and ethylene were constructed for olive SE pretreated
respectively with 0.25M, 0.5M and 0.75M of sucrose, mannitol or sorbitol. The most
successful pretreatment enhanced glutathione reductase (GR) activity compared to
controls, whereas superoxide dismutase (SOD) catalase and guaiacol peroxidase
activities remained relatively unchanged. Superoxide dismutase activity was higher
in SE pretreated with sucrose, compared to those pretreated with mannitol and sor-
bitol. Hydrogen peroxide (H2O2) was enhanced in SE cultures pretreated with sorbitol
and sucrose, compared to those exposed to mannitol. Ethylene and hydroxyl radical
(OH) levels increased in SE pretreated with mannitol and sorbitol. Pretreatments
did not change SE profiles of ThioBarbituric Acid Reactive Substances (TBARS) and
Schiff’s bases, although the use of these assays is cautioned because of interference
from cryoprotective sugars and polyols.
These studies suggest that oxidative stress influences recovery of cryopreserved
olive SE; they concur with findings obtained for Ribes shoot meristems using encap-
sulation/dehydration (Johnston et al., 2007. Plant Science 172, 524–534). Biochemical
401
profiling using markers of oxidative stress and antioxidants may facilitate evidence-
based decisions regarding cryopreservation protocol improvement in diverse plant
species and germplasm types. This study has been informed by whole plant research
of O. europaea in which it has been observed that olive trees develop antioxidant and
compatible solute protection strategies in nature. As this occurs as part of a natural
adaptation to drought stress (Sofo et al., 2008. Hydrology and Earth Systems Sciences
12, 293–301) it may be exploited to aid olive cryostorage.Thus, optimal pretreatments
increase tolerance to cryopreservation by enhancing endogenous levels of antioxi-
dants, proline and protective sugars; their manipulation in vitro simulates the natural
adaptations of olive trees exposed to drought stress and thereby improves cryostor-
age outcomes.
Acknowledgements: Prof E Rugini for plant cultures; technical support, IRD,
Montpellier, France; Universities of Derby and Abertay Dundee, UK; Erica Benson
for a Visiting Research Fellowship at the University of Derby.
Conflict of interest: None.
Source of funding: EU COST Action 871; CRYMCEPT QLK5CT-2002-01279.
doi:10.1016/j.cryobiol.2010.10.132
129. Exploring standard preanalytical coding for environmental biospecimens using algal
cryobanks as a case study. Erica E. Benson * 1,2, Fotini Betsou 2,3, Keith Harding 1,3,
1
Damar Research Scientists, Drum Road, Cuparmuir, Fife, Scotland KY15 5RJ, UK,
2
Integrated Biobank of Luxembourg, 6, rue Ernest Barblé, L-1210, Luxembourg,
3
Biospecimen Science Working (sub)-Group, ISBER, 9650 Rockville Pike, Bethesda,
MD 20814-3993, USA
The International Society for Biological and Environmental Repositories (ISBER)
Working Group on Biospecimen Science has recently developed a Standard Preanalyt-
ical Code (‘SPREC’) for clinical specimens (Betsou et al., in press. Cancer Epidemiology,
Biomarkers and Prevention. This biospecimen ‘barcode’ provides information about
the preanalytical processing of samples, which takes place between specimen collec-
tion, (which can be inclusive of cryostorage) to the point of their experimental use in
analysis and research. Biospecimen science has emerged in the healthcare sector to
clarify the contribution of the cellular and molecular alterations that occur in biospec-
imens because of their biobank process history, rather than intrinsic differences
attributed to specimens per se. The rationale being, the more precision afforded to
recording a sample’s process history the more accurate and explicit the information
that can be gained from biospecimen research, especially when it involves different
collaborating institutions. Thus, tracing and understanding the impacts of preanalyt-
ical variables is important as they can potentially affect sample quality and lead to
experimental variation that may be difficult to attribute and identify. The clinical
SPREC was designed to meet the increasing demands of the end users of healthcare
biobanks, especially those using sensitive analyses, undertaking molecular/omics
research and engaging in large-scale consortia projects which require parity across
sample processing procedures.
In support of ISBER’s environmental remit, the objective of this presentation is to
explore the wider potential for adapting and adopting the clinical SPREC biospecimen
barcode in non-healthcare settings. As a case study, algal cryobanks have been chosen
because they comprise different biorepository models which offer diverse holdings of
organisms to different types of end users. Algal culture collections residing in, and
servicing the environmental research sector offer the advantage that biospecimen col-
lectors, curators and end users can come under the operational jurisdiction of the
repository. The clinical SPREC (designated version 01) is a simple, seven element-long
code, formulated using existing laboratory management tools and technical protocols
(e.g. sample preparation, centrifugation, cryoprotection, freezing and storage regime).
Thus, a similar approach is used to test the feasibility of adapting the SPREC in algal
cryobanks, including its scope for being applied to some technical processes before
and after cryostorage. The range of the SPREC is based on the qualifying criteria that
all its parts are under the control of the biorepository adopting the system, and that it
is easy and intuitive to use. This presentation considers the benefits of adopting pre-
analytical codes, balanced against the risks of instigating over-complicated quality
assurance tools in repositories with limited resources. The SPREC has considerable
applicability as it can be implemented as a simple ‘‘low tech” hand written record,
or a digitally formatted ‘supermarket inventory barcode’. The versatility and potential
for using the SPREC in non-clinical biobanks and their communities of practice will be
presented with a view to developing quality and traceability tools that enable collab-
orations across algal research infrastructures.
Conflict of interest: None.
Source of funding: Integrated Biobank of Luxembourg.
Reference
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doi:10.1016/j.cryobiol.2010.10.133