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Abstracts: CryoLetters 24 (3), 2003

CryoLetters 24, 133-134 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Barry Fuller



CryoLetters 24, 135-142 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


T. Hoshino*1, M. Kiriaki1 and T. Nakajima2

1 Research Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan
2 National Agricultural Research Center for Kyushu Okinawa Region (KONARC), 2421 Suya, Nishigoshi, Kikuchi, Kumamoto 861-1192, Japan


The low temperature basidiomycete, Coprinus psychromorbidus, produced three kinds of thermal hysteresis proteins (THPs) in the extracellular space. Molecular mass of purified fungal THPs were approximately 23 kDa, respectively, however those fungal THPs had different N-terminal amino acid sequences. Those fungal THPs formed ice crystals resembling "Stone Age arrow head". These observations indicate that fungal THPs did not form hexagonal ice crystals to inhibit its growth.

Keywords: antifreeze protein, Coprinus psychromorbidus, ice morphology, low temperature basidiomycete, snow mold fungi, thermal hysteresis, thermal hysteresis protein



CryoLetters 24, 143-148 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Kazuhito Kajiwara1*, Kazuyoshi Yabe and Takusei Hashitani2

Department of Biosciences, Teikyo University of Science and Technology, 2525 Yatsusawa, Uenohara-machi, Kitatsuru-gun, Yamanashi 409-0193, JAPAN
1Present address; School of Bionics, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, JAPAN
2Home 15-3-601, Shimoochiai 4-chome, Shinjuku-ku, Tokyo 161-0033, JAPAN


A volume change method for measuring crystal densities is described. It allows the densities of unstable hydrated crystals at room temperature to be determined, by measurements of volume changes during the solidification of aqueous solutions. NaCl·2H2O, KCl, MgSO4·12H2O and K2HPO4·6H2O were measured by the method and their densities are 1.61±0.02, 1.99 ± 0.05, 1.45 ± 0.01 and 1.75 ± 0.02 g ml-1 respectively. Data of NaCl·2H2O and KCl are in good agreement with the previously reported values.

Keywords: Density, Volume change, Unstable hydrated crystals, Solidification of aqueous solutions



CryoLetters 24, 149-160 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Stéphane Dussert1*, Florent Engelmann1, 2 and Michel Noirot1

1 UMR DGPC, Institut de recherche pour le développement (IRD), 911 Av. d'Agropolis, BP 64501, F34394 Montpellier Cedex 5, France. (email address for S.D.:
2 International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


A simple method, based on the binomial distribution, is proposed to calculate the probability of recovering at least one (or any other fixed number of) plant(s) from a cryobank sample using four given parameters: the percentage of plant recovery observed from a control sample, pobs, the number of propagules used for this control, n1, the number of propagules in the cryobank sample, n2, a chosen risk for the calculation of a confidence interval for the observed plant recovery, Using this method, it is possible to assess the number of propagules which should be rewarmed immediately after freezing in order to estimate the plant recovery percentage as a function of the total number of propagules available. It also allows the calculation of the minimum plant recovery percentage to ensure that the probability to recover at least one (or A, with A>1) plant(s) is higher than a fixed probability level, as a function of the control and the cryobank sample sizes. Reciprocally, once the plant recovery percentage has been estimated, it is possible to assess the minimum size of the cryobank sample to obtain a probability to recover at least one (or A, with A>1) plant(s) higher than some fixed level.

Keywords: cryobank, recovery, sample size, germplasm, cryopreservation, probability



CryoLetters 24, 161-170 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A numerical Study of cell behaviour in a ternary solution during the freezing process

Dawei Luo1,2, Liqun He2, Shuxia Cheng2, Jianping Yu1 and Dayong Gao1

1Department of Mechanical Engineering, University of Kentucky, Lexington, KY, U.S.A. 40506
2 department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei, Anhui, China


Using a continuum model for multi-component phase change system, the freezing of cell suspension in a ternary solution, H2O-NaCl-CPA (cryoprotective agent) inside a flat bag is investigated numerically in this study. The temperature and phase change history, intracellular water loss, and the volume change of the cells at different locations inside cell suspension are calculated. Numerical results reveal that although the sample boundary is cooled at a constant rate, different locations inside the sample experienced different temperature changes and cooling rates. The highest cooling rates occur at internal locations. The cell volume change is location-dependent.

Keywords: phase change heat conduction; cell model; nucleation mechanism; cryoinjury



CryoLetters 24, 191-200 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Selection of overwintering microhabitats used by the Arctic woollybear caterpillar, Gynaephora groenlandica

Valerie A. Bennett1*, Richard E. Lee, Jr.1, Jennifer S. Nauman2, and Olga Kukal3

1Department of Zoology, Miami University, Oxford, OH  45056 USA;
2Biology Department, Millikin University, Decatur, IL 62522 USA;
3Atlantic Low Temperature Systems, Ltd., Halifax, NS B2Y 4L3 Canada;
*Corresponding author & current address from August 15 2003: Biology Department, Colorado State University-Pueblo, Pueblo, CO 81001 USA


In extreme environments such as the High Arctic, climatic conditions challenge physiological tolerance of insects resulting in prolonged dormancy and extended life cycles. Therefore, the selection of suitable microhabitats for overwintering is crucial. At two field sites on Ellesmere Island, we located hibernacula (silk overwintering structures) used by the Arctic woollybear caterpillar, Gynaephora groenlandica (Wöcke) (Lepidoptera: Lymantriidae) during their 11 month dormancy.  All hibernacula found were anchored to the base of rocks and were not associated with vegetation. Rocks may function to absorb solar radiation and re-radiate heat, accelerating localized snowmelt, thus allowing caterpillars to emerge as early as possible in spring to begin foraging.  Temperatures experienced by G. groenlandica in hibernacula throughout the winter were well within their physiological tolerance range. During late summer aestivation, hibernaculum temperatures were similar to, but more stable than, nearby soil surface temperatures.  Lower maximum daily hibernaculum temperatures during the warmest month (July) may reduce metabolic rates and provide some energy savings. Since hibernacula were not randomly distributed around rocks, this suggests that G. groenlandica key in on some feature of the hibernaculum site.  The northeast orientation of hibernacula at Eastwind Lake corresponds to the leeward side of rocks.  Therefore wind patterns may be important in hibernaculum site selection. Other potential cues that may guide selection of hibernacula sites remain unclear: no seasonal changes in preference for light or soil moisture were observed between active and dormant caterpillars, and thermotaxis could not be distinguished from thigmotaxis.

Keywords: hibernaculum, Lepidoptera, microhabitat, temperature, behavior



CryoLetters 24, 171-180 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Evaluation of Damage in pacific oyster (crassostrea gigas) spermatozoa before and after cryopreservation using comet assay

Jin-Chywan Gwo*, Chen-Yen Wu, Wu-Shu Peter Chang1 and Hsien-Yu Cheng2

Department of Aquaculture, Taiwan National Ocean University, Keelung, Taiwan 20224.    
1 Institute of Environmental Health Sciences, National Yangming University, Taipei, Taiwan
2 Department of Biology, Chinese Culture University, Taipei, Taiwan 110.


We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10% DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length%, tail area%, and tail intensity%) were higher than the oyster sperm protected with 10%DMSO-artificial sea water after freezing and thawing.  Only tail length%, tail area%, and tail intensity% were increased significantly after cryopreservation.  The tail length % was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development.  This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.

Keywords: DNA, freeze, mollusks, sperm, oyster, comet assay



CryoLetters 24, 181-190 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Neelam Sharma1* and Bindu Sharma2

1Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110012, India
 2Present address –Jubilant Bioinformatics Ltd., Bangalore
*Corresponding author; email


The cryopreservation of shoot tips of Picrorhiza kurroa Royle ex Benth (IC 266698), an endangered medicinal plant of India was investigated. Shoot tips (about 1 mm in length) excised from four-week-old proliferating shoot cultures were precultured on MS medium supplemented with various osmotica before dehydrating with PVS2 solution at 0ºC. The dehydrated shoot tips were directly immersed in LN2. Following cryopreservation, and after rapid rewarming at 45ºC, shoot tips were quickly washed with 1.2 M sucrose solution and then plated on solidified shoot culture medium. Shoot tips were successfully cryopreserved by vitrification, when they were precultured on medium supplemented with 5% DMSO at 4ºC for two days before dehydrating in PVS2 for 10-20 minutes at 0ºC. Average survival in terms of normal shoot formation after 4 wks of plating was about 20% without callus formation. Cold hardening of shoot cultures for four weeks at 4ºC significantly improved the survival and shoot regeneration of cryopreserved shoot tips to 70% and 35%, respectively.

Key words: cryopreservation, endangered plant, Picrorhiza kurroa, medicinal plant, vitrification, PVS2


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