Abstracts: CryoLetters 33 (4), 2012

CryoLetters is a bimonthly, international journal for low temperature science and technology



Volume 33, No. 4 July/August 2012

ISSN 0143-2044



Reverse altitudinal cline in cold hardiness among Erebia butterflies
Pavel Vrba, Martin Konvička and Oldřich Nedvěd




Cryopreservation of Lomandra sonderi (asparagaceae) shoot tips using droplet-vitrification
Akshay Menon, Bryn Funnekotter, Anja Kaczmarczyk,
Eric Bunn, Shane Turner and Ricardo L. Mancera




‘Personalisation’ of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects
Haeng-Hoon Kim and Sheong-Chun Lee




Multi-decadal survival of an antarctic nematode, Plectus murrayi, in a -20°c stored moss sample
H. Kagoshima, K. Kito, T. Aizu, T. Shin-i, H. Kanda,
S. Kobayashi, A. Toyoda, A. Fujiyama,Y. Kohara,
P. Convey and H. Niki




Proteomic expression of microfungal ripening starter Geotrichum candidum submitted to cold stress is strain-dependent: studies using 2D-DIGE technology and SameSpotsTM software analysis
Ghalia Missous, Bouachanh Thammavongs,
Virginie Dieuleveux, Maryline Houssin, Joël Henry
and Jean-Michel Panoff




The effect of cryopreservation on genomic stability in strains of the fungus Trichoderma
Richard Broughton, Alan G. Buddie, David Smith
and Matthew J. Ryan




The effects of trehalose and sucrose on frozen spermatozoa of Yunnan semi-fine wool sheep during a non-mating season
Guo Bo Quan, Qiong Hua Hong, Qing Yong Shao,
Hong Yuan Yang and Shuai Shuai Wu




Long-term preservation of chilled canine semen using vitamin C in combination with green tea polyphenol
Manita Wittayarat, Taichi Kimura, Risa Kodama,
Zhao Namula, Kaywalee Chatdarong,
Mongkol Techakumphu, Yoko Sato, Masayasu Taniguchi
and Takeshige Otoi





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CryoLetters 33 (4), 251-258 (2012)
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Pavel Vrba, Martin Konvička and Oldřich Nedvěd*

Faculty of Science, University of South Bohemia, Branisovska 31, 37005 Ceske Budejovice, Czech Republic and Institute of Entomology, Academy of Sciences of the Czech Republic, Branisovska 31, 37005 Ceske Budejovice, Czech Republic.
*Corresponding author


There is strong evidence for a shifting of range boundaries by many temperate butterfly species to higher altitudes and latitudes. Climate change represents a potential threat to mountain fauna. Nevertheless, information on ecophysiological limits of individual species is scarce.  We studied the lower thermal limits of four species representing the prevailingly mountain Holarctic butterfly genus Erebia. We measured the cold tolerance of hibernating larvae, namely the supercooling point (SCP) and the lower lethal temperature (LLT). Three mountain species were freeze avoiding, with various levels of SCP (–8 to –22°C), and LLT close to SCP. The only exception was lowland E. medusa, whose caterpillars were freeze tolerant with LLT (–21°C) slightly below its SCP (–17°C). Surprisingly, LLT was highest in the alpine E. tyndarus and lowest in E. medusa inhabiting lower altitudes with higher mean winter temperatures. We explain the observed reversed altitudinal cline in cold hardiness by the buffering function of snow cover in the hibernacula of caterpillars that is strong at high mountains but irregular, unpredictable and thus unreliable in lowlands.

Keywords: Alpine habitats, butterfly ecology, climate change, snow cover



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CryoLetters 33 (4), 259-270 (2012)
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Cryopreservation of Lomandra sonderi (Asparagaceae) shoot tips using droplet-vitrification

Akshay Menon1,2, Bryn Funnekotter1,2, Anja Kaczmarczyk1,2,
Eric Bunn2,3, Shane Turner2,3 and Ricardo L. Mancera1,4*

1Western Australian Biomedical Research Institute, Curtin Health Innovation Research Institute, School of Biomedical Sciences, Curtin University, GPO Box U1987, Perth WA 6845, Australia.
2Botanic Gardens and Parks Authority, Fraser Avenue, West Perth WA 6005, Australia.
3School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley WA 6009, Australia.
4School of Pharmacy, Curtin University, GPO Box U1987, Perth WA 6845, Australia.
*Corresponding author email:


A cryopreservation protocol was developed for Lomandra sonderi (Asparagaceae), an endemic plant of southwest Western Australia used for mine site restoration. Thermal analysis of L. sonderi shoot tips using differential scanning calorimetry was used to detect the formation of ice in shoot tips and consequently allowed optimisation of the time of incubation in plant vitrification solution 2 (PVS2), which attempted to minimise phytotoxicity of, and excessive dehydration by, its cryoprotective components. Sugar pretreatments did not improve survival. Use of a loading solution containing 2 M glycerol and 0.4 M sucrose prior to incubation in PVS2 improved survival of control shoot tips. Preconditioning at 20/-1°C day/night alternating temperature with a 16 h photoperiod or at a constant 5°C temperature with a 12 h photoperiod both significantly improved both control shoot tip survival and post-cryopreservation survival. Shoot tips that recovered from liquid nitrogen immersion were successfully re-established as actively growing in vitro plantlets.

Keywords: Lomandra sonderi, cryopreservation, droplet-vitrification, thermal analysis, differential scanning calorimetry, cold acclimation



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CryoLetters 33 (4), 271-279 (2012)
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Haeng-Hoon Kim and Sheong-Chun Lee*

Department of Well-being Resources, Sunchon National University, Suncheon 540-742, Korea.
*Corresponding author email:


Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions. This paper proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions. The proposed standard protocol consists of a progressive preculture with 0.3 M sucrose for 31 h and with 0.7 M for 17 h, loading with vitrification solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 20 to 40 min, dehydration with vitrification solutions A3-90% (37.5% glycerol + 15% DMSO + 15% EG + 22.5% sucrose) for 10 to 30 min or B1-100% (PVS3) for 40 to 120 min at room temperature, cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40°C) unloading solution (0.8 M sucrose) and further unloading for 20 to 60 min, depending on size and permeability of the materials. Using this systematic approach we can identify whether the material is tolerant or sensitive to chemical toxicity and to the osmotic stress of dehydration with vitrification solutions, thus revealing which is the main barrier in solution-based vitrification methods. Based on the sensitivity of samples we can design a droplet-vitrification procedure, i.e. preculture, loading, dehydration with vitrification solutions, cooling and rewarming. Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.

Keywords: chrysanthemum, garlic, Kalopanax callus, potato, Rubia hairy roots, standard protocol



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CryoLetters 33 (4), 280-288 (2012)
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H. Kagoshima1,2*, K. Kito3, T. Aizu2, T. Shin-i2, H. Kanda4,
S. Kobayashi1, A. Toyoda2, A. Fujiyama2,Y. Kohara2,
P. Convey5 and H. Niki1,2

1Transdisciplinary Research Integration Center, Research Organization of Information and Systems (ROIS), Toranomon 4-3-13, Minato-ku, Tokyo 105-0001, Japan
2National Institute of Genetics, ROIS, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
3Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
4National Institute of Polar Research, ROIS, Midori-machi 10-3, Tachikawa, Tokyo, 190-8518, Japan
5British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 0ET, United Kingdom
*Corresponding author  email:


It is not clear for how long Antarctic soil nematodes might tolerate freezing. Samples of the Antarctic moss, Bryum argenteum, were collected on 1 October 1983 at Langhovde, Soya coast, eastern Antarctica and were stored at -20°C. After 25.5 years of storage, living nematodes were recovered from the samples and were identified as Plectus murrayi by morphological examination and nucleotide sequencing of ribosomal RNA loci. The nematodes can grow and reproduce in a water agar plate with bacteria (mainly Pseudomonas sp.) cultured from the moss extract. They showed freezing tolerance at -20°C and -80°C and their survival rate after exposure to -20°C, but not -80°C, was increased if they were initially frozen slowly at a high sub-zero temperature. They also showed some ability to tolerate desiccation stress.

Keywords: Plectus murrayi, nematode, freezing, desiccation, long-term storage, Antarctica



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CryoLetters 33 (4), 289-298 (2012)
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Proteomic expression of microfungal ripening starter Geotrichum candidum submitted to cold stress is strain-dependent: studies using 2D-DIGE technology and SameSpotsTM software analysis

Ghalia Missous1,2,* Bouachanh Thammavongs1,
Virginie Dieuleveux2, Maryline Houssin2,
Joël Henry3 and Jean-Michel Panoff1,4

1Unité des Microorganismes d'Intérêts Laitier et Alimentaire, IBFA – IFR 146 ICORE, Université de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen cedex, France.
2Laboratoire Départemental F. Duncombe, 1 rte Rosel, 14053 Saint Contest, France.
3UMR 100 IFREMER Physiologie et Ecophysiologie des Mollusques Marins – IFR 146 ICORE, Université de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen cedex, France.
4Plateau de cryobiologie – IFR 146 ICORE, Université de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen cedex, France.
*Corresponding author email:


Geotrichum candidum is a micro-fungus widely used as a ripening starter in cheese making. In anthropogenic environments such as dairy industries, this microorganism is subjected to many environmental and technological stresses including low temperature exposure. Our aim was to study the proteomic response of G. candidum to cold stress using a comparative proteomic approach by two-dimensional Differential In Gel Electrophoresis (2D DIGE). This technique consists on the labeling of proteins by specific fluorescent dyes (CyDyesTM). The results, obtained with G. candidum cells subjected to cold temperature, show significant proteomic patterns differences compared with the standard conditions. Furthermore, this biochemical response seems strain specific. 2D DIGE technology combined with SameSpotsTM software analysis support these results through an important statistical validity. The comparative studies in a single gel, using two different fluorescent CyDyes (Cy3 and Cy5), lead to proteins differentiation. Selected spots were treated and analyzed by mass spectrometry.

Keywords: cold, stress, 2D DIGE, Geotrichum candidum, microfungi, starter.



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CryoLetters 33 (4), 299-306 (2012)
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The effect of cryopreservation on genomic stability IN strainS of the fungus Trichoderma

Richard Broughton1, 2, Alan G. Buddie1, David Smith1
and Matthew J. Ryan*1

1CABI Europe-UK, Bakeham Lane, Egham Surrey, TW20 9TY
2Royal Holloway, University of London, Egham, Surrey, TW20 0EX


Within Biological Resource Collections, the successful long-term storage of fungal cultures is essential because of their scientific and potential commercial value. Preservation procedures such as cryopreservation have traditionally been used to ensure genomic stability due to the suspension of metabolic activity at ultra-low temperatures. Genomic integrity is important with regards to conservation, as changes in the genome may compromise production of a desired metabolite, enzyme or activity. To evaluate cryopreservation as a conservation protocol, genomic integrity was assessed in five strains of the economically important fungus Trichoderma. Two polymerase chain reaction (PCR) fingerprinting techniques commonly used for molecular studies of fungi were applied. Three genetic polymorphisms were detected amongst replicates post preservation, indicating that even robust, standardised preservation cryopreservation methodologies can sometimes induce genomic change. However, the low number of polymorphisms suggests that cryopreservation is a reliable method for organism storage over long periods of time.

Keywords: biomarkers, Biological Resource Centres, genomic stability, PCR fingerprinting, filamentous fungi



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CryoLetters 33 (4), 307-317 (2012)
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Guo Bo Quan, Qiong Hua Hong*, Qing Yong Shao,
Hong Yuan Yang and Shuai Shuai Wu

Yunnan Animal Science and Veterinary Institute, Qinglongshan, Jindian, the Panlong County, Kunming 650224, Yunnan province, China
*Corresponding author e-mail: Mrs Qiong Hua Hong, M.D.

Statement of funding: This study was funded by the National Wool Caprine Industrial Technology System (Grant No. CARS-40).


To improve the quality of frozen spermatozoa of Yunnan semi-fine wool sheep, the protective effect of trehalose and sucrose on frozen ram spermatozoa during a non-mating season was evaluated and compared in this study. Briefly, following collection by electric stimulation, equilibration at 5℃æ following dilution with the freezing extender, and pre-freezing in liquid nitrogen vapor, the ram spermatozoa were frozen in liquid nitrogen. After thawing, viability, motility, acrosome status, membrane integrity, and phosphatidylserine (PS) distribution was determined using a computer-assisted spermatozoa analysis system and flow cytometry. The data indicated disaccharide can improve the quality of frozen ram spermatozoa. With a trehalose concentration of 100mM, the post-thaw viability and motility (80.56%±6.89% and 46.07%±5.84%) of ram spermatozoa were significantly more than those of ram spermatozoa frozen with no disaccharide (65.46%±18.96% and 34.62%±9.32%, P<0.05). However, the effect of sucrose on the viability, motility, and moving velocity of ram spermatozoa was similar to that of the control group (P>0.05). Compared with sucrose, trehalose can significantly increase the motility of frozen ram spermatozoa (P<0.05). In addition, addition of trehalose or sucrose can efficiently protect the acrosome of frozen spermatozoa. Moreover, when the concentration of trehalose or sucrose was 100mM, the protective effect of trehalose or sucrose on the membrane integrity and PS distribution was significantly higher than that of the control group (P<0.05). However, the protective effect of trehalose on viability, moving velocity, acrosome status, membrane integrity, and PS distribution of frozen ram spermatozoa was similar to that of sucrose (P>0.05). In conclusion, the protective effect of trehalose on frozen sheep spermatozoa is superior to that of sucrose. Addition of 100mM of trehalose in the freezing extenders can improve the post-thaw quality of ram spermatozoa with respect to viability, motility, and linear velocity. Moreover, presence of disaccharide can protect acrosome and membrane of frozen sheep spermatozoa.

Keywords: sheep; spermatozoa; cryopreservation; trehalose; sucrose; freezing



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CryoLetters 33 (4), 318-326 (2012)
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