CryoLetters 24 (6), 2003

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

CryoLetters 24, 341-346  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

cracking in a vitrification solution DURING COOLING OR WARMING does not effect growth of cryopreserved mint shoot tips

Leigh E. Towill and Remi Bonnart

USDA-ARS National Center for Genetic Resources Preservation, Fort Collins, CO 80521, USA


No obvious decrease in viability or in the ability of mint shoot tips to develop into a shoot occurred during vitrification when the external glass cracked upon either cooling or warming. Samples within semen straws did not show a decrease in survival over three cycles of cooling and warming either in the presence or absence of cracking. No physical defects were visible in treated shoot tips. Cracking of the external glass formed from PVS2 did not obviously influence shoot tip survival in mint species.

Keywords: repetitive cooling, Mentha, fracturing, cryopreservation



CryoLetters 24, 347-358  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Jacob G. Bundy1, Hans Ramløv2 and Martin Holmstrup3*

1 Biological Chemistry, Imperial College of Science, Technology and Medicine, Division of Biomedical Sciences, Faculty of Medicine, Sir Alexander Fleming Building, London SW7 2AZ, UK.
2 Roskilde University, Department of Life Sciences and Chemistry, PO Box 260, DK-4000 Roskilde, Denmark.
3 National Environmental Research Institute, Department of Terrestrial Ecology, PO Box 314, Vejlsøvej 25, DK-8600 Silkeborg, Denmark. * Email:


Individuals of the freeze-tolerant earthworm, Dendrobaena octaedra, and four freeze-intolerant earthworm species (Dendrodrilus rubidus, Aporrectodea icterica, A. caliginosa, and A. longa) were frozen at -2C. Control earthworms were exposed to +2C. 1H nuclear magnetic resonance spectroscopy-based metabolic profiling in combination with multivariate pattern recognition methods (metabonomics) was used to produce a cross-species comparison. Several biochemical changes were detected as a result of freezing in all worm species, including an increase in relative free alanine concentrations, and an apparent conversion of adenosine to inosine. It was also possible to determine a number of biochemical changes that were unique to the freeze-tolerant species, D. octaedra. The most obvious difference was that, although all species showed an increase in glucose concentrations, the increase was largest in D. octaedra, and was coupled with a concomitant decrease in glycogen. This confirms that – like previously studied freeze-tolerant earthworm species – tolerance is effected by rapid glucose production from glycogen reserves. An additional difference noted was that succinate increased in all species on freezing, but the increase was least in D. octaedra. Furthermore there was no lactate accumulation in D. octaedra, whereas three of the other four species accumulated lactate. This indicates that anoxic metabolism was lowest in the freeze-tolerant species.

Keywords: earthworms; freeze-tolerance; nuclear magnetic resonance spectroscopy; glucose



CryoLetters 24, 359-364 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Donor Liver Preservation and Stimulation of Adhesion Molecules : Cold Preservation Initiates the Cascade

Van Hoang, Mohamed El Wahsh, Patrice Butler, Keith Rolles, Brian Davidson and Barry Fuller*

University Department of Surgery & Liver Transplant Unit, Royal Free Hospital and University College Medical School, London NW3 2QG, UK.


Background: Pro-inflammatory adhesion molecules (including ICAM-1, E-Selectin) are important in clinical hepatic ischaemia / reperfusion injury. The initiating factors remain to be fully elucidated.
Methods: Needle biopsies taken during and after donor liver preservation were studied in 28 cases by immuno-histochemical methods. Human responder cell cultures were exposed to the effluent cold storage solution after liver preservation in a further 10 transplants.
Results: Increased ICAM-1 staining was seen at the end of cold storage compared to baseline (P<0.01), whilst reperfusion caused further increase (P<0.001). The storage solutions stimulated ICAM-1 and E-Selectin expression in responder cells in vitro as a bio-assay (P<0.01 in each case).
Conclusions: Liver harvesting and cold storage can initiate adhesion molecule expression even before reperfusion in the recipient.

Keywords: donor liver preservation; storage solution; adhesion molecules; ICAM-1



CryoLetters 24, 365-374 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development

J. Kopeika, E. Kopeika1, T. Zhang*, D. M. Rawson

Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, LU1 5DU, UK
1Institute for Problems of Cryobiology and Cryomedicine, National Academy of Science of Ukraine, 23 Pereyaslovskaya Str, Kharkov 61015, Ukraine


The process of sperm cryopreservation consists of several steps: equilibration of sperm in cryoprotectant medium, freezing of sperm to subzero temperatures, low temperature storage and thawing of the sperm suspension. It has been shown that cryopreservation can cause some damage to the genetic material of cells although the mechanism and significance of these changes are still unknown. The aim of this work was to study the effect of cryoprotectant equilibration process on genetic damage of Loach (Misgurnus fossilis) sperm, using embryo survival as an indicator. Decrease in embryo survival after the 20th stage is generally believed to result from the failure in the genome function of embryos. In the first set of the experiments, Loach sperm were equilibrated in cryoprotectants Me2SO, ethylene glycol, methanol and glycerol (0.6, 1.2, 2.5 M) for 60 min at 10C. The effect of cryoprotectant equilibration on sperm was evaluated based on the survival of embryos derived from cryoprotectant treated sperm. Embryo survival was evaluated at the following stages: 7th, 14th, 17th, 20th, 23rd, 26th, 31st, 34th, 35th, 36th and 37th. Cryoprotectants at concentrations greater than 1.2 M had significant effect on the survival of the embryos after the 20th stage. The effect of glycerol was the most significant with 64.82.4% of embryos survival compared to 77.02.4% for control. Me2SO treatment also effects embryo survival significantly. Possible mechanisms of the genetic instability of cryoprotectants are discussed.

Keywords: Loach (Misgurnus fossilis), sperm, embryo survival, cryoprotectant, toxicity, genome damage.



CryoLetters 24, 375-380  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Yu-Jin Hao1,2* Yun-Jiang Cheng1 and Xiu-Xin Deng1*

1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, P. R. China
2(present address) Department of Plant, Cell and Environment, National Institute of Fruit Tree Science, Fujimoto 2-1, Tsukuba, Ibaraki 305-8605, Japan (E-mail:
*Authors for correspondence.


The conservation of transgenic materials is very important, paticularly for their potential future use in crop development. In this study, transgenic callus cultures of ‘Newhall’ navel orange (Citrus sinensis Osbeck) were cryopreserved by a vitrification method. Transgenic calluses survived cryopreservation and recovered under normal culture conditions. The results of PCR (Polymerase Chain Reaction) amplification, Southern blotting and SSCP (Single-Strand Conformation Polymorphism) assay showed that the GUS gene was still maintained in the genome of callus cultures recovered from cryopreservation. X-Gluc staining further indicated GUS gene expression in callus cultures recovered from cryopreservation.

Key words: vitrification, GUS gene, PCR,  SSCP, Southern blotting



CryoLetters 24, 381-388 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

IMPORTANCE OF explant size and origin and of PRECONDITIONING treatments for Cryopreservation of GARLIC shoot apices BY VITRIFICATION

Hyung-Jin Baek1, Haeng-Hoon Kim1*, Eun-Gi Cho1 , Young-Am Chae2 and Florent Engelmann3, 4

1National Institute of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707, Korea (email for H.H.K.:
2Department of Agronomy and Research Center for New Biomaterials in Agriculture, Seoul National University, Suwon 441-744, Korea.
3Cirad-Flhor, TA 50/PS4, Boulevard de la Lironde, 34398 Montpellier cedex 5, France. (present address).
4International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


This paper investigates the effect of the origin and size of the explants employed and of the preconditioning (cold acclimation, preculture) and loading treatments on survival and regeneration of cryopreserved garlic shoot apices using vitrification with the PVS3 vitrification solution. Both the origin and size of explants had a significant effect on regeneration of cryopreserved apices. Higher regeneration was generally observed with apices excised from bulbs and bulbils, followed by cloves, and those originated from larger propagules regrew more rapidly. Smaller apices (1.5 or 3.0 mm in diameter) displayed higher regeneration than large ones (4.5 mm in diameter). Cold acclimation at 5 °C of apices before freezing had no positive effect on regeneration after cryopreservation. Preculture of apices at 10 or 23 °C for more than 3 days had a detrimental effect on regeneration. The optimal sucrose concentration in the preculture medium was 0.3-0.5 M. Loading apices for 30 or 60 min at 23 °C in medium containing 2 M glycerol + 0.4 M sucrose or 1 M glycerol + 0.8 M sucrose had no effect on regeneration after cryopreservation, in comparison with apices cryopreserved without loading treatment. Under optimal conditions, regeneration of cryopreserved apices sampled from large cloves was above 90 %.

Keywords: Allium sativum L.; shoot apex; preconditioning treatment; explant size; explant origin; vitrification.



CryoLetters 24, 389-396  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cold Storage and Cryopreservation of Hops (Humulus L.) shoot cultures through application of standard Protocols

Barbara M. Reed1*, Nese Okut2, Jeff D’Achino1, Lee Narver1, and Jeanine DeNoma1

1U.S. Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333. Email:
2Assistant Professor, Yuzuncu Yil Universitesi, Ziraat Fakultesi 65080 Van, Turkey


The USDA-ARS National Clonal Germplasm Repository (NCGR) stores the global diversity of Humulus for the US Plant Germplasm System as trellised plants in a field genebank. In vitro storage and cryopreservation are now considered excellent ways to provide medium and long-term storage for plant collectionsDeveloping a new cryopreservation or cold storage protocol for every accession or genus of large multi-crop collections can be a very time consuming and long-term activity. We propose that standard cold storage and cryopreservation techniques used for other temperate crop genera would be successful for additional crops with few modifications.  This study was initiated to determine if a large collection of hops germplasm could be successfully stored with techniques developed for unrelated genera. In this study we characterized the response of diverse Humulus genotypes to in vitro storage under low light at 4 C following techniques used for strawberry and mint plants, and cryopreservation in liquid nitrogen by slow cooling with a pear protocol. The average storage time without transfer for the 70 genotypes evaluated was 14 + 3.5 months with a range of 6 to 26 months.  Mean recovery of cryopreserved shoot tips of accessions with 1-wk cold acclimation was 41% + 18 and increased to 54% + 13 with 2-wk cold acclimation.  This demonstrates that application of a well-tested standard technique can provide a quick start for storing additional germplasm collections. 

Keywords: cold acclimation, cryopreservation, germplasm, Humulus, in vitro storage, liquid nitrogen, slow cooling.



CryoLetters 24, 397-412  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Abstracts of TEMP2003

International Symposium on Animal and Plant Cold Hardiness

August 10 - 14, 2003, České Budějovice, Czech Republic


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