Abstracts: CryoLetters 30 (4), 2009

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



Volume 30, No. 4 July/August 2009

ISSN 0143-2044



Cryopreservation of in vitro shoot apices of Glehnia littoralis – a medical plant
Keishu Otokita, Daisuke Kami, Takashi Suzuki and Masahiko Suzuki




Two-step rapid freezing method: estimation of membrane hydraulic permeability in erythrocytes at temperature exposure stage
N. V. Repin




Cryopreservation of dormant buds from diverse Fraxinus species
G. M. Volk, R. Bonnart, J. Waddell, and M. P. Widrlechner




Thermal analysis and cryopreservation of seeds of australian wild Citrus species (rutaceae): Citrus australasica, C. inodora and C. garrawayi
Kim N. Hamilton, Sarah E. Ashmore and Hugh W. Pritchard




Cryopreservation of shoot tips of Trichilia emetica, a tropical recalcitrant-seeded species
Dalia Varghese, Patricia Berjak and N.W. Pammenter




Development of alternative loading solutions in droplet-vitrification procedures
Haeng-Hoon Kim, Yoon-Geol Lee, Sang-Un Park, Sheong-Chun Lee, Hyung-Jin Baek, Eun-Gi Cho and Florent Engelmann




Apoptosis in human ovarian tissue after conventional freezing or vitrification and xenotransplantation
Gohar Rahimi, Vladimir Isachenko, Plamen Todorov, Samir Tawadros, Peter Mallmann, Frank Nawroth and Evgenia Isachenko




Editorial – Changes at CryoLetters; Future Meeting – Cryo 2010





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CryoLetters 30 (4), 244-250 (2009)
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Keishu Otokita*, Daisuke Kami, Takashi Suzuki and Masahiko Suzuki

Department of Horticultural Science and Landscape Architecture, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan.
*Corresponding author e-mail:


Cryopreservation was examined as a practical method for preserving the genetic resources of Glehnia littoralis Fr. Schm. a nearly exterminated medical plant. In vitro shoot apices were successfully cryopreserved using vitrification and encapsulation-dehydration. In vitrification, regrowth of apices loaded for 60 min with PVS2 (66.7 ± 6.7%) was preferable to loading with PVS3 (50.0 ± 5.8%). With encapsulation-dehydration, the best regrowth (43.3 ± 3.3%) was achieved when the moisture content in the beads was reduced to 19% by drying with silica gel for 6 h. Increased regrowth of shoot tips cryopreserved by encapsulation-dehydration resulted from the addition of 10-4 M acetylsalicylic acid to the loading solution (86.7 ± 3.3%).

Keywords: acetylsalicylic acid, cryopreservation, encapsulation-dehydration, Glehnia littoralis, vitrification



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CryoLetters 30 (4), 250-261 (2009)
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N. V. Repin

Department of Cryomorphology, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 23, Pereyaslavskaya str., Kharkov, 61015, Ukraine.
Corresponding author e-mail address:


The processes, occurring at the stage of temperature exposure under two-step freezing of human  and hibernating ground squirrel erythrocytes were studied by electron-microscopic method of freeze-fracturing. For human erythrocytes the time of intracellular crystal dissolution in human erythrocytes made 30 min and this value was shown to be slightly depending on temperature within -20°C ÷ -35°C range.

Erythrocyte membrane permeability for water within negative temperature range has been estimated. It was noted that the value of water membrane permeability coefficient obtained theoretically, taking into account experimental values of time of dissolution for intracellular ice crystals and cell dehydration, is quite well consistent with the reported data and equals to about 0.5x10-13N-1m 3sec-1 for human erythrocytes and 10-13N-1m3 sec-1 for hibernating ground squirrel  cells.

Under two-step freezing a temperature arrest results in a qualitative change of water exchange process between cell and its environment, and the exposure duration may serve as the measure for cell dehydration.

According to the data of NMR “paramagnetic doping” and freeze-fracturing the erythrocyte membrane of hibernating ground squirrels is more permeable for water than human one.

Keywords: erythrocyte, two-step freezing, intracellular crystal recrystallisation, freeze-fracturing method, hydraulic permeability, dehydration.



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CryoLetters 30 (4), 262-267 (2009)
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Cryopreservation of dormant buds from diverse Fraxinus species

G. M. Volk1*, R. Bonnart1, J. Waddell1, and M. P. Widrlechner2

1USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521, USA
2USDA-ARS North Central Regional Plant Introduction Station, Iowa State University, Ames, IA 50011, USA
* Corresponding author e-mail:


Ash (Fraxinus) is an economically important tree genus in the landscape industry, as well as a key component of North American forests, especially in the North Central United States and adjacent regions in Canada.  In recent years, the Emerald Ash Borer beetle (Agrilus planipennis) has significantly threatened the survival of native North American Fraxinus species. A dormant-bud cryopreservation technique has been developed as a method to conserve specific clones of ash. Dormant buds of three ash species were successfully cryopreserved when desiccated on their stem sections to 30% moisture content (w/v) and then cooled at rates of either -1ºC/h or -5ºC/day to either -30 or -35ºC before immersion in liquid nitrogen vapor (LNV). Stem sections were removed from LNV, warmed, and rehydrated, and their buds grafted onto rootstocks to evaluate survival. Recovery percentages ranged from 34 to 100% after LNV exposure and were dependent upon accession and cooling rate. The cryopreservation methods proposed herein can complement seed-collection efforts aimed at conserving diversity, supplementing ex situ genebank and botanic-garden collections.

Keywords: ash, genetic resources, genebank, Fraxinus pennsylvanica, Fraxinus mandshurica, Fraxinus chinensis



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CryoLetters 30 (4), 268-279 (2009)
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Kim N. Hamilton1,2*, Sarah E. Ashmore2 and Hugh W. Pritchard3

1Botanic Gardens Trust Sydney, Mount Annan Botanic Gardens, Mount Annan, NSW 2567 Australia
2Centre for Forestry and Horticultural Research, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, QLD 4111 Australia
3Seed Conservation Dept., Royal Botanic Gardens Kew, Wakehurst Place, West Sussex RH17 6TN, UK
*Corresponding author email:


We investigated the relationship between the thermal transitions in seeds, cryopreservation and geographical origin for the rare and threatened northern Australian Citrus species, Citrus inodora and C. garrawayi, and southeastern Australian species  C. australasica, which is cultivated as a ‘bushfood’. Thermal analysis of phase transitions in cotyledon tissue revealed differences between species in the melt onset temperatures of in vivo seed oils, suggestive of differences in the proportion of saturated fatty acids. These differences appeared to be associated with geographic gradient, i.e. an increased mean onset temperature of lipid melt coincided with latitude (N NSW / SE QLD Australia to N QLD) and climatic zone (warm subtropical to hot tropical) of the natural distribution range. In addition, the thermal transitions of seed oils corresponded to the temperature limit for germination.  Tolerance to cryopreservation was demonstrated in all three species after drying, with a mean germination of 75±2, 71±7 and 42±12% for C. australasica, C. inodora and C. garrawayi, respectively, when dried below the unfrozen water content (WCu) determined for each species. All three species have edible fruits and seed cryopreservation now offers an alternative strategy for the long-term ex situ conservation of this valuable germplasm.

Keywords: crop wild relative, differential scanning calorimetry (DSC), desiccation, germination, natural distribution, seed oils



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CryoLetters 30 (4), 280-290 (2009)
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Dalia Varghese, Patricia Berjak* and N.W. Pammenter

School of Biological and Conservation Sciences, University of KwaZulu-Natal, Westville Campus, Durban, 4001 South Africa.
*Corresponding author e-mail:


This paper reports the successful cryopreservation of shoot tips of Trichilia emetica, a tropical tree species producing recalcitrant seeds. Preculture of shoot tips on MS medium with 0.7 M sucrose or with 0.3 M sucrose + 0.5 M glycerol followed by cryoprotection with a mixture of glycerol and DMSO or with PVS2 was crucial for successful recovery following cryostorage. Three cooling rates were applied to assess the effects on post-thaw regrowth of shoot tips. Slow cooling of the shoot tips (WC 1.24 g g-1 DW) precultured on medium with 0.3 M sucrose + 0.5 M glycerol and cryoprotected with PVS2 resulted in high shoot production (71%). Subsequent to relatively faster cooling, only 38% of the shoot tips developed shoots. Ultra-rapid cooling with PVS2 resulted in callus formation with 55% regrowth. We report one of the very few successful attempts to cryopreserve explants alternative to zygotic axes of tropical tree species producing recalcitrant seeds.

Keywords: cooling rates, cryopreservation, preculture, shoot tips, vitrification

Abbreviations: BAP: 6-benzylaminopurine; DMSO: dimethyl sulphoxide; DW: dry weight; GA3: gibberellic acid; LN: liquid nitrogen; MS: Murashige & Skoog medium; PVS2: plant vitrification solution 2; WC: water content; WPM: woody plant medium



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CryoLetters 30 (4), 291-299 (2009)
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Haeng-Hoon Kim1*, Yoon-Geol Lee1, Sang-Un Park2, Sheong-Chun Lee3, Hyung-Jin Baek1, Eun-Gi Cho1 and Florent Engelmann4,5

1National Academy of Agricultural Science, RDA, Suwon 441-707, Korea.
2Chungnam National University, Daejon, 305-764, Korea.
3Sunchon National University, Suncheon, 540-742, Korea.
4Institut de recherche pour le développement (IRD), UMR DIAPC, BP 64501, 34394 Montpellier cedex 5, France.
5Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Corresponding author email:


In plant vitrification protocols, the loading treatment, which involves treating the explants with a moderately concentrated cryoprotectant solution, precedes dehydration of explants with highly concentrated vitrification solutions in order to reduce the toxicity which can be induced by their direct exposure to such highly concentrated solutions. This study aimed at developing alternative loading solutions composed of mixtures of glycerol and sucrose at various concentrations. Differential scanning calorimetry runs of loading solutions and of loaded and dehydrated explants were performed to assay thermal events occurring during cooling and warming. These loading solutions were applied to two model species, viz. garlic and chrysanthemum which were cryopreserved using a droplet-vitrification procedure. The loading treatment proved to be beneficial to both garlic and chrysanthemum and increased recovery of cryopreserved explants. However, response to the loading solutions tested varied between the two model species employed: with garlic, all the loading solutions had a similar effect, whereas survival of chrysanthemum shoot tips was significantly influenced by the composition of the loading solution employed. A loading solution comprising 1.9 M glycerol and 0.5 M sucrose was the most effective. The loading treatment may thus act as an osmotic stress neutralizer and/or induce the physiological adaptation of tissues and cells, including membranes, to both dehydration and freezing.

Keywords: chrysanthemum, garlic, droplet-vitrification, loading solution.



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CryoLetters 30 (4), 300-309 (2009)
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Gohar Rahimi1, Vladimir Isachenko1,2, Plamen Todorov3, Samir Tawadros4, Peter Mallmann1, Frank Nawroth5 and Evgenia Isachenko1,2*

1Department of Obstetrics and Gynaecology, University of Cologne, Kerpener Str.43, 50931 Cologne, Germany
2Section of Gynaecological Endocrinology and Reproductive Medicine, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany
3Institute of Biology and Immunology of Reproduction, Tzarigradsko shosse 73, 1113 Sofia, Bulgaria
4Department of Internal Medicine, University of Cologne, Kerpener Str.43, 50931 Cologne, Germany
5Endokrinologikum Hamburg, Centre of Hormone and Metabolic Diseases, Reproductive Medicine and Gynaecological Endocrinology, Lornsenstraße 22767 Hamburg, Germany
*Corresponding author  e-mail:


One of the new emerging techniques to preserve reproductive potential of cancer patients is cryopreservation of ovarian fragments prior to medical treatment and their retransplantation after healing. In order to investigate and compare apoptosis in human ovarian tissue after conventional (“slow”) freezing and vitrification, we used a xenograft model in which conventionally frozen, vitrified and fresh non treated human ovarian tissue pieces were subcutaneously transplanted in SCID mice. The tissue samples were weekly, during four weeks, recovered from scarified SCID mice. The apoptosis was examined by immunohistochemical staining with the anti-caspase-3 antibody. There was a significant difference between the amount of apoptotic cells in cryopreserved ovarian tissue independent from mode of cooling compare to the control. The ovarian tissue after vitrification showed a significantly higher amount of apoptotic cells, than in slow frozen. The results obtained after comparative study of two different cryopreservation methods show that vitrification of human ovarian tissue could become a practice-relevant alternative to slow cryopreservation only after further improvement.

Keywords: apoptosis, human ovarian tissue, slow freezing, vitrification, transplantation

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