CryoLetters 25 (3), 2004

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

CryoLetters 25, 155-160  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A simple method to adjust Cooling rates for supercooling point determination

M. A. Carrillo1, N. Kaliyan2, C. A. Cannon1*, R. V. Morey2 and W. F. Wilcke2

1Department of Entomology, University of Minnesota, Saint Paul, MN 55108 USA.
* Tel: 612-625-4798; Fax: 612-625-5299;
2 Department of Biosystems and Agricultural Engineering, University of Minnesota, Saint Paul, MN 55108 USA.


A simple method to obtain predetermined constant cooling rates for insect supercooling point (SCP) determination is described. A transient heat transfer equation was used to design polystyrene cubes of different sizes to yield constant rates of cooling at their centers when held at a constant surrounding temperature. Cubes of 0.29 0.29 0.29 m and 0.19 0.19 0.19 m were found to produce cooling rates of ca. -0.5 and -1oC min-1, respectively, from 0 to -40oC. The observed temperature variations at the geometrical center of the cubes were similar to those predicted by the equation. Temperature plots showed a nearly constant rate of cooling. Supercooling points of Tribolium castaneum (Herbst) at different stages of development were recorded using polystyrene cubes. These SCPs compared favorably with published values. This method of obtaining cooling rates is economical, flexible, and allows for multiple simultaneous SCP measurements.

Keywords: Cooling rates, cold hardiness, supercooling point, Tribolium castaneum



CryoLetters 25, 161-166  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Are mummy characteristics reliable indicators of diapause and cold tolerance in the parasitoid wasp Aphidius rhopalosiphi (Braconidae, Aphidiinae) ?

M.A. Legrand1*, C. Salin1, A. Langer2 and T. Hance1

1*Université catholique de Louvain, Centre de Recherche sur la Biodiversité, Unité d’Ecologie et de Biogéographie, 5 Place Croix du Sud, 1348 Louvain-la-Neuve, Belgique.
2 Dr Alain Langer, Haute-Fagnes-Eifel Nature Park, Centre Nature de Botrange, B-4950, Robertville, Belgium.


Many insect species have evolved different overwintering survival strategies such as cold tolerance and/or diapause. This study investigated the relationship between Aphidius rhopalosiphi mummy colour and cold tolerance and diapause. Mummy colour was insufficient to discriminate diapausing from non-diapausing individuals. This phenotypic character seems to reflect environmental conditions rather than direct developmental time and cocoon thickness (identification criteria of diapause). There is, however, a relationship between cold tolerance and mummy colour. Dark mummies exhibited significantly higher water content, survival at low temperature and lower supercooling point values than pale mummies. Mummy colour in Aphidius rhopalosiphi seems to be a phenotypic indicator of the cold tolerance.

Keywords: diapause, cold tolerance, mummy colour, Aphidiinae, Aphidius rhopalosiphi



CryoLetters 25, 167-176  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Daisuke Tanaka1, Takao Niino2, Kanji Isuzugawa3, Takashi Hikage4 and Matsuo Uemura1,5*

1United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
2Gene Bank, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan
3Yamagata Prefectural Horticultural Experimental Station, Sagae 991-0043, Japan
4Ashiro Floricultural Development Center, Ashiro 028-7533, Japan
5Cryobiosystem Research Center, Iwate University, Morioka 020-8550, Japan
* Corresponding author (


Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars/lines. Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0% and 73.7% for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.

Keywords: cryopreservation, encapsulation-vitrification, Gentiana, shoot apices, vitrification



CryoLetters 25, 177-186  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of embryogenic cultures from mature Quercus suber trees using vitrification

Silvia Valladares1, Mariano Toribio2, Cristina Celestino2 and Ana M. Vieitez1*

1Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apdo. 122, 15080 Santiago de Compostela, Spain. E-mail:
2Instituto Madrileño de Investigación Agraria y Alimentaria (IMIA), Finca “El Encín”, Apdo. 127, 28800 Alcalá de Henares (Madrid)


Recent progress in somatic embryogenesis from selected mature trees of Quercus suber, has led to a demand for maintenance of a large number of selected embryogenic lines. To facilitate the management of this material a protocol for the long-term storage of this germplasm should be defined. This study reports on the use of a simple vitrification procedure for the successful cryopreservation of three cork oak embryogenic lines. High embryo recovery levels (88-93%) were obtained by first preculturing 2-4 mg clumps of two or three globular embryos on semisolid medium containing 0.3 M sucrose for three days, followed by incubation in PVS2 vitrification solution at 0ºC for 60 min before direct immersion in liquid nitrogen. The mean number of embryos produced per explant was significantly greater for cryostored embryos than for untreated stock cultures, but the productivity of the latter was recovered in subsequent subcultures of the material produced by cryostored embryos. The germination and plant regeneration rates achieved by cultures derived from cryostored embryos, around 60%, were similar to those of non-cryopreserved stock cultures. 

Keywords: cork oak, cryogenic storage, cryopreservation, somatic embryogenesis, Quercus suber, vitrification.



CryoLetters 25, 187-194  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


R T Cook. Kingston University, Kingston upon Thames, Surrey KT1 2EE, United Kingdom


Cold hardiness of ectotherms has been widely studied in arthropods, but there is a more limited literature on the survival of molluscs at low temperatures.  A number of intertidal species have been examined in detail, but terrestrial molluscs have largely been overlooked until recently. This paper reports results of laboratory experiments to evaluate the cold hardiness of the terrestrial slug, Deroceras reticulatum.  The mean supercooling point (SCP) rose from -4.2C in summer to -3.6C in winter.  The SCP that caused 50% mortality (LSCP50) remained constant at -4.7 to -4.8C in both seasons, but slugs were able to survive the frozen state for longer in winter (LD50 of 31.8 minutes compared with 17.0 minutes in summer).  Slug survival at freezing temperatures was prolonged to at least five hours when placed on a moist, absorbent substrate. D. reticulatum exhibits partial freeze tolerance, with an increased survival in winter. The results are discussed in relation to the slugs' natural environment.

Keywords: cold hardiness, Deroceras, freezing, molluscs, slugs, supercooling



CryoLetters 25, 195-204  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Hitoshi Obata*, Naomi Muryoi, Hidehisa Kawahara and Ayako Nishiyama

Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35, Yamate-cho, Suita-shi, Osaka, 564 –8680, Japan.


The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30oC to 12oC, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30oC to 12oC, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30oC to 12oC) and a non-cold acclimation culture (30oC), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83% identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30% at 5.0 µg/ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70oC for 30 min, but had no activity after treating at 100oC. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.

Keywords: Pantoea agglomerans NBRC12686; freezing tolerance; cold acclimation; cryoprotective activity; uridine phosphorylase; ribose-1- phosphate



CryoLetters 25, 205-212  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


A.S. Popov *, E.V. Popova, T.V. Nikishina and  G.L. Kolomeytseva1

Institute of Plant Physiology of Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia. E-mail: (A.S. Popov)
1Main Botanical Garden of Russian Academy of Sciences, Moscow


The development of juvenile plants of hybrid Bratonia orchid in vitro after seed storage in liquid nitrogen and the effect of nutrient medium composition on protocorm multiplication and plant regeneration were investigated. Cryopreservation did not inhibit the germination rate of seeds. Protocorms derived from cryopreserved seeds developed faster than protocorms from control (unfrozen) seeds during the first 45 days. But during further culturing, this tendency was not retained and finally protocorms from cryopreserved seeds had the same size as control ones. There were no significant differences in leaf number and shoot length between juvenile plants derived from unfrozen and cryopreserved seeds. We found that among four tested media liquid Morel medium was the most preferable for protocorm multiplication, and liquid МS medium with half-strength macronutrients was the best one for the development of juvenile plants.

Keywords: Seed cryopreservation, orchids, Bratonia



CryoLetters 25, 213-217  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


W. Harris1, P.T. Lynch2*, A.J. Hargreaves1 and P.L.R. Bonner1

1School of Science, The Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, UK.
2Division of Biological Sciences, University of Derby, Kedleston Road, Derby, DE22 1GB, UK.
* To whom correspondence should be addressed (e.mail:


Helianthus tuberosus cell suspension cultures were subjected to cryopreservation 24h preculture treatments with 0.5M sucrose or mannitol. Extracts were assayed for transglutaminase activity and the level of α-tubulin tyrosination. There was a significant reduction (compared with the non-precultured controls) in transglutaminase activity and α-tubulin tyrosination state after mannitol preculture treatment, whereas sucrose preculture treatment produced no significant effect. The results suggest that reduced levels of transglutaminase activity and α-tubulin tyrosination are associated with a lack of post-thaw recovery observed following mannitol preculture treatment of cell culture suspensions. These activities may represent useful molecular markers of the success of preculture treatments in cryopreservation protocols.

Keywords: Transglutaminase, post-thaw regrowth, cytoskeletal proteins, Helianthus tuberosus, tubulin.



CryoLetters 25, 219-226  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of Garlic (Allium sativum L) using Plant Vitrification Solution 2

Gayle M. Volk*, Nicholas Maness and Kate Rotindo

USDA/ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA
*To whom correspondence should be addressed; Email:


Most cryopreservation procedures are optimized using a small number of germplasm accessions.  We classified the garlic (Allium sativum L.) accessions that were selected for our studies based on genotype as identified using amplified fragment length polymorphism markers.  Although recovery was variable, shoots regenerated from a broad range of the accessions after cryo-exposure. Garlic shoot tips were excised from cloves, surface sterilized, and placed on media at 5oC for 2 days prior to cryopreservation. Shoot tips were then treated with sucrose-glycerol for 20 minutes, plant vitrification solution 2 (PVS2; 15% w/v ethylene glycol, 15% w/v DMSO, 30% w/v glycerol, 13.7% w/v sucrose) at 0oC, and then plunged on foils into liquid nitrogen slush.  Explants were recovered in 1.2 M sucrose for 20 minutes and then plated onto Gamborg’s B5 medium containing ྟ-naphthaleneacetic acid (NAA) and 6-(ྙ࿠ྙ -dimethylallylamino purine) (2-iP).  Our results demonstrate that genotypically diverse accessions of garlic can be successfully cryopreserved.

Keywords: cryopreservation, garlic, shoot tip, media, PVS2



CryoLetters 25, 227-234  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Norio Murase1*, Satoru Abe2, HiroshiTakahashi2, Chihiro Katagiri3, Takumi Kikegawa4

1Department of Biotechnology, School of Science & Engineering, Tokyo Denki University, Hatoyama, Hiki-gun, Saitama 350-0394, Japan.
2Faculty of Engineering, Gunma University, Maebashi 371-8510, Japan.
3Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan.
3Institute of Materials Structure Science, High Energy Accelerator Research Organization, Tsukuba 305-0801, Japan.


Ice crystallisation in crosslinked dextran (Sephadex) gels was studied by the method of two-dimensional X-ray diffraction (XRD) in combination with the simultaneous measurement of differential scanning calorimetry (DSC). With a Sephadex G25 gel where an exotherm due to ice crystallisation is observed in the DSC rewarming trace, it was indicated by the XRD pattern that small ice crystals less than ~ 10 μm in diameter are readily formed during freezing, and that the endothermic trend prior to the exotherm is not due to the glass transition but due to the melting of the small ice crystals. Moreover, the diffraction pattern observed with frozen Sephadex gels depended on the density of crosslink indicating that ice crystals of different size and dimension are formed in the gels.

Keywords: Polymer gels, crosslinked dextran (Sephadex), ice crystallisation during rewarming, two-dimensional XRD, DSC



CryoLetters 25, 235-236  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Letter to the Editor


Gary C. Packard
Mary J. Packard
Department of Biology,
Colorado State University,
Fort Collins,
CO 80523-1878,




International Symposium On The Environmental Physiology Of Ectotherms And Plants (ISEPEP2005), Roskilde, Denmark, 11 – 16 July, 2005

Society For Low Temperature Biology, 40th Annual Meeting, London, 9 – 10 September, 2004

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