Abstracts: CryoLetters 25 (5), 2004

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

CryoLetters 25, 307-310 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Jae-Shin Kang1 and James A. Raymond

Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154 USA
1Permanent address: Korea Polar Research Institute, Korean Ocean Research and Development Institute (KORDI), Ansan, South Korea


Antarctic sea ice diatoms produce ice-binding proteins (IBPs) that are strong inhibitors of the recrystallization of ice. Their function may be to reduce cell damage in the frozen state. We show here that an IBP from the diatom Navicula glaciei Vanheurck also has the ability to reduce freeze-thaw damage to red blood cells and that the effect may be due to its ability to inhibit recrystallization of ice.

Keywords: Cryopreservation, diatom, recrystallization inhibition, ice-active substance



CryoLetters 25, 311-322 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Takashi Kamata1 and Matsuo Uemura1,2*

1United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
2Cryobiosystem Research Center, Iwate University, Morioka 020-8550, Japan
* Correspondence author (E-mail:


Accumulation of sugars, amino acids and glycinebetaine in leaf tissues during cold acclimation was simultaneously monitored and compared in three wheat cultivars that have different freezing tolerance. Freezing tolerance was the order of cv. Norstar (NO) > cv. Chihokukomugi (CH) ≥ cv. Haruyutaka (HA). During cold acclimation, there was a significant increase in osmotic concentration in the three cultivars. The increase was largely due to the increase in soluble sugars and the extent of the increase was the greatest in NO and the least in HA. While there was a considerable increase in glucose, fructose and sucrose during the first week of cold acclimation, the increase in raffinose occurred only after the second week. The total sugar content was the order of NO > CH > HA after 4 weeks of cold acclimation. Proline increased in all cultivars after 1 week of cold acclimation but a prolonged cold acclimation resulted in different profiles: no further increase occurred in HA while an additional increase occurred in other two cultivars. In all three cultivars, a noticeable increase of glycinebetaine occurred only after the second week of cold acclimation with the amount being the order of NO > CH > HA. It is concluded that a substantial part of the increase in osmotic concentration during cold acclimation was due to the increase in sugars, but the extent of the contribution of each compatible solute is cultivar-specific and can be associated with the degree of the maximum freezing tolerance attainable.

Keywords: cold acclimation, compatible solutes, wheat, freezing tolerance, osmotic concentration



CryoLetters 25, 323-334 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Leigh E. Towill 1*, Philip L. Forsline 2, Christina Walters1, John W. Waddell1 and Julie Laufmann1

1 USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA.
2 USDA-ARS Plant Genetic Resources Unit, Cornell University, Geneva, NY 14456, USA


Cryopreservation using a winter vegetative bud method is being applied to the Malus collection maintained in the field at the USDA-ARS Plant Genetic Resources Unit, Geneva NY. Winter hardy materials are sent to the USDA-ARS National Center for Genetic Resources Preservation, Fort Collins, CO, for processing.  To date 1915 accessions, representing 30 species and 16 interspecific hybrids, have been tested. The NCGRP minimum standard for cryopreservation is 40% viable buds, as determined by grafting. For M. x domestica 95% of the accessions tested have been cryopreserved. For species other than M. x domestica, 83% have met the criterion.   Eight lines were collected, cryopreserved and recovered through grafting each year.  Data from this set showed an affect of year and cultivar on success.  There was no strong relationship between viability after cryopreservation and phylogeny.  For North American species success after cryopreservation was related to geographical origin.  

Keywords:   genetic resources, germplasm preservation, liquid nitrogen, long-term storage, apple



CryoLetters 25, 335-340 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A simple ice nucleation spectRometer

David A. Wharton1, *, Jodi S. Mutch2, Peter W. Wilson3, Craig J. Marshall2 and Miang Lim4

Departments of Zoology1, Biochemistry2, Physiology3 and Food Science4, University of Otago, P.O. Box 56, Dunedin, New Zealand.
*Corresponding author


The construction of a simple ice nucleation spectrometer is described. It uses 10 µl droplets loaded into glass capillary tubes which are then inserted into an aluminium holder. Each holder takes six capillary tubes surrounding a central thermocouple. Four holders are placed into a cooling block, cooled by fluid from a programmable refrigerated circulator, and the thermocouples interfaced to a computer to record temperatures. Freezing of each sample is detected by an exotherm on the temperature recording, with 24 samples recorded per run. The spectrometer was tested using deionized water, an extract from a New Zealand alpine cockroach and an extract of lawn grass. The cockroach extract is estimated to contain about 103 more nucleators, active at 5°C, than the grass extract.

Keywords: ice nucleation spectrometer, exotherm, freezing, Celatoblatta quinquemaculata, grass



CryoLetters 25, 341-352 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Barbara M. Reed1, Irina Kovalchuk2, Svetlana Kushnarenko2, Andreas Meier-Dinkel3, Katja Schoenweiss3, Stanislaw Pluta4, Krystyna Straczynska4, and Erica E. Benson5

1*USDA-ARS National Clonal Germplasm Repository, 33447 Peoria Rd, Corvallis, OR 97333-2521 Email:
2Institute of Plant Physiology, Genetics and Bioengineering, 45 Timiryazev Str., Almaty, 480090, Republic of Kazakhstan
3Lower Saxony Forest Research Institute, Department of Forest Gene Conservation, Forstamtstrasse 6, D-34355 Staufenberg-Escherode, Germany
4Research Institute of Pomology and Floriculture Skiernievwice, Poland
5School of Contemporary Sciences, University of Abertay, Dundee, Dundee, Scotland KY15 5RJ, UK


Cryopreservation of plant tissues in liquid nitrogen is now used for long-term conservation of vegetatively-propagated crops. Development of standard techniques for cryopreservation is important to the international plant-conservation community for successful implementation of storage protocols in diverse and internationally dispersed laboratories. Evaluation of the critical points of each preservation technique will greatly assist in developing and validating internationally-used cryopreservation protocols. The goals of this project were to assess critical points of two major cryopreservation techniques (PVS2 vitrification and encapsulation dehydration) during their transfer to international laboratories; analyze post-storage viability for each technique and location; and develop recommendations based on the assessments and data from the participating laboratories.  Investigators from Germany, Kazakhstan, Poland and UK participated in a 2-week training workshop in cryopreservation methods after which the techniques were tested in the home laboratories of the participants. After one-year site visits by the technology trainers identified critical points in the protocols. Critical points were identified as 1) Cryogenic (cryoprotection, LN exposure, rewarming); 2) Non-cryogenic (plant health status, pre- and post-storage culture); 3) Operational (skills transfer, training, interpretation of procedures); 4) Facility (growth room, ambient conditions, media preparation, equipment). The most critical factors in all laboratories were culture health, operator skills and experience, and clarification of the technical details of the procedures.  Final results showed that correction of critical factors improved the post-storage recovery in all the laboratories.

Keywords: Meristem, shoot tip, Ribes, liquid nitrogen, germplasm



CryoLetters 25, 353-362 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Lin Gui, Zhong-Shan Deng, Jing Liu*

Cryogenics Laboratory, P. O. Box 2711, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China


In this article, a minimally invasive injection method to enhance the addition of cryoprotective agent into the target biological tissues or organs to be cryopreserved was studied. Compared with the traditional treatment through immersion and permeation, direct injection could be much more effective and faster. To illustrate the benefit of this approach, a mathematical model for the addition of cryoprotective agent was established and experiments were performed to test the differences given by the two methods. Both the theoretical and the experimental results demonstrate that the minimally invasive injection method for enhancing the addition of cryoprotective agent is a promising substitution for the commonly used immersing method.

Keywords: Cryoprotective agent; minimally invasive injection; cryopreservation; enhanced addition; porous media



CryoLetters 25, 363-374 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Recovery of cryopreserved embryogenic culturesof Maritime pine - Effect of cryoprotectant and suspension density

Liliana Marum1, Catarina Estêvão1, M. Margarida Oliveira1,2*, Sara Amâncio3, Lucília Rodrigues3 And Célia Miguel1

1instituto de Biologia Experimental e Tecnológica/ Instituto de Tecnologia Química e Biológica, Quinta do Marquês, 2784-505 Oeiras, Portugal
2 Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal, Campo Grande, 1749-016 Lisboa, Portugal
3Centro de Botânica Aplicada à Agricultura do Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal
*to whom correspondence should be addressed
E-mail:   Fax: +351 21 4433644


Embryogenic cultures were obtained from seeds of open-pollinated maritime pine trees representing part of a breeding population. The aim of the present study was to develop and optimize a protocol for cryopreservation of Pinus pinaster somatic embryogenic tissue. The density of the embryogenic suspension and the type of pre-treatment significantly affected the recovery of cryopreserved embryogenic cultures, as evaluated by their survival and regrowth rate. An initial density of the suspension culture of 250 mg/ml improved the regrowth rate of the embryogenic lines. Pre-treatment with maltose 0.4 M significantly increased the regrowth rate when compared to the other tested carbohydrates. Also, the addition of Me2SO in a mixture of PEG 4000 and sucrose (PSD solution), instead of Me2SO alone, at the same final concentration, was clearly beneficial for recovery of cryopreserved tissues. This improved method for the cryopreservation of P. pinaster embryogenic of cultures allowed the successful recovery of 97% of the lines stored in liquid of nitrogen.

Keywords: Pinus pinaster, somatic embryogenesis, cryopreservation, cryoprotectant, suspension density.

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