Abstracts: CryoLetters 29 (3), 2008

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

CryoLetters 29(3), 181-188 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Esther E. Uchendu1 and Barbara M. Reed2*

1Department of Horticulture, ALS 4017, Oregon State University, Corvallis, OR, 97331, USA.
2National Clonal Germplasm Repository, United States Department of Agriculture, Agricultural Research Service, 33447 Peoria Rd, Corvallis, OR, 97333-2521, USA.
Corresponding author: Barbara M. Reed e-mail:


This study was designed to determine the response of diverse mint genotypes to three commonly used cryopreservation techniques. Four mints [Mentha x piperita nothosubsp. citrata (Ehrh.) Briq.; M. canadensis L.; M. australis R. Br, and M. cunninghamii Benth] were cryopreserved using three protocols: controlled rate cooling (CC), encapsulation dehydration (ED) and PVS2 vitrification (VIT). Regrowth of mint species following controlled rate cooling (93%) was significantly (P<0.0001) better than encapsulation dehydration (71%) and vitrification (73%). All four genotypes responded well to the controlled rate cooling protocol but there was some variability with the other two protocols. Genotype specific response to the individual protocols showed that there were significant differences in the recovery of Mentha x piperita nothosubsp. citrata and M. australis with CC > VIT > ED. There were also significant differences in the recovery of M. cunninghamii and M. canadensis, with CC and ED significantly better than VIT. Regrowth of the shoot tips of these mints ranged from 60% to 95% for all but one treatment. The overall results of this study compare favorably to other techniques. These improved results may be due to a combination of favorable growth conditions, cold acclimation and recovery medium. Controlled rate cooling was the most successful technique for the storage of these diverse mint genotypes; however recovery of shoot tips from VIT and ED was high and these techniques could also be used for cryogenic storage of mint germplasm.

Keywords: cold acclimation, controlled rate cooling, encapsulation dehydration, in vitro, vitrification



CryoLetters 29(3) 189-198 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

The development and limits of freezing tolerance in Acer pseudoplatanus fruits across Europe is dependent on provenance

Matthew I. Daws and Hugh W. Pritchard*

Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, U.K.
*Corresponding author: Hugh W. Pritchard e-mail:


The effects of fruit maturity, at the time of natural dispersal, on subsequent desiccation tolerance and sub-zero storage was investigated in three lots of Acer pseudoplatanus (sycamore) collected from northern to southern Europe. Fruits from the native plant distribution range in Italy had significantly higher desiccation tolerance (0.16 g H2O g-1 DW) than those from England (0.30) and Norway (0.50), confirming that the maximum potential desiccation tolerance in sycamore exceeds that of the recalcitrant type. In contrast, the unfrozen water content varied only slightly between seedlots, but systematically reduced with development (0.35 to 0.27 g H2O g-1 DW). Maximum survival (60% fruit germination) of seven days sub-zero temperature storage coincided with drying the Italian fruit lot to c. 0.2 g H2O g-1 DW followed by holding at -20 °C, above the onset temperature for freezing, or at -196°C (liquid nitrogen). Fruit survival was much lower in the Italian fruits when held at this water content and -70°C, and in all other combinations of water content, temperature and fruit lot provenance. As the risk of nucleation in partially dried fruits held at -20°C is high, we recommend sycamore fruits are cryopreserved for long-term conservation.

Keywords: Acer pseudoplatanus, type II seed, differential scanning calorimetry, desiccation tolerance, seed maturity



CryoLetters 29(3), 199-208 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Artur Kowalczyk

Department of Poultry Breeding, Wroclaw University of Environmental and Life Sciences, Chelmoñskiego 38c, 51-630 Wroclaw, Poland


Changes in the morphology and fertilising ability of Japanese quail spermatozoa were studied after semen dilution, equilibration and freezing-thawing process in order to determine the optimal diluent, cryoprotectant and the freezing-thawing method.

Subsequent stages of quail semen cryopreservation caused significant decline in spermatozoa morphology and their ability to fertilise the ovum. Semen dilution with Lake’s extender alone reduced the number of morphologically normal spermatozoa and decreased their fertilising ability. Dimethylacetamide (DMA) was the least detrimental but equilibration of quail spermatozoa with this cryoprotectant caused further decline in the number of morphologically normal cells. However, despite these changes, after artificial insemination with semen equilibrated with DMA 25.8% of fertile eggs were obtained. Further loss in the number of normal spermatozoa was observed following the freezing-thawing process. Of the two investigated freezing-thawing methods, the “rapid” rate (60°C/min) appeared less detrimental to spermatozoa morphology and their ability to fertilize the ovum than the “slow” rate. Also the number of sperm holes appearing in the inner perivitelline layer and the number of spermatozoa trapped in the outer perivitelline layer of the ovum was higher after the ‘rapid’ than the ‘slow’ freezing-thawing procedure. Nevertheless, both rates did not yield any fertile eggs.

Keywords: quail, semen, cryopreservation, morphology, fertilisation



CryoLetters 29(3), 209-216 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Daisuke Kami*, Maki Uenohata, Takashi Suzuki and Katsuji Oosawa
Department of Horticultural Science and Landscape Architecture, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan
*Corresponding author e-mail:


In vitro shoot apices of black chokeberry (Aronia melanocarpa Elliott) were successfully cryopreserved utilizing three methods, namely vitrification, encapsulation-vitrification and encapsulation-dehydration. Encapsulation-dehydration and encapsulation-vitrification, however, seem preferable to vitrification, since the highest respective survival levels of apices (71.1 ± 2.2% and 77.8 ± 4.4%) by both methods were higher than that (60.0 ± 3.9%) by vitrification. In encapsulation-dehydration, the highest survival was achieved when the moisture content of beads was reduced to 19% by drying with silica gel for 6 h. In the present study, it was shown that adding 1.0 M glycerol to beads and loading solution during encapsulation-dehydration resulted in high survival (91.7 - 95.0%) regardless of lines and polyploids of black chokeberry.

Keywords: black chokeberry, cryopreservation, encapsulation-dehydration, encapsulation-vitrification, glycerol



CryoLetters 29(3), 217-228 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


N. Bakradze*, E. Kiziria, V. Sokhadze, and S. Gogichaishvili

E. Andronikashvili Institute of Physics, 6 Tamarashvili str, Tbilisi, 0107 Georgia
E. Kiziria -,
V. Sokhadze -,
Sh. Gogichaishvili -
*Correspondence author: N. Bakradze e-mail:


Results are presented of a water phase transition study in plantain (Plantago major L.) roots, which were used as a model system to research the peculiarities of water crystallization and ice melting processes in complex heterogeneous biological systems. It was confirmed that water in such systems is crystallized in two clearly distinguished temperature ranges: -10 to -25 °C and -25 to -45°C. These water fractions are conditionally attributed to extracellular (-10 to -25°C) and intracellular (-25 to -45°C) solutions. A possible explanation is given for such significant supercooling of the intracellular solution. The values of osmotic pressures of extra- and intracellular solutions were determined according to ice melting curves. It is noted that the intracellular solution, which crystallized at lower temperatures, had a lower osmotic pressure.

Keywords: DSC, plant root, water supercooling, intracellular crystallization, osmotic pressure.



CryoLetters 29(3), 229-242 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of sugarcane somatic embryos

Marcos E. Martinez-Montero1*; Julia Martinez1 and Florent Engelmann2,3

1University of Ciego de Avila, Bioplantas Centre, Car. a Moron km 9, CP 69450, Ciego de Avila, Cuba (Email:
2Institut de recherche pour le d éveloppement (IRD), UMR DIA-PC, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France. (Email:
3Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Corresponding author: e-mail:


In this paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25°C, treatment with the PVS2 solution for 20-40 min at 0°C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55%.

Keywords: somatic embryos, sugarcane, vitrification, encapsulation-vitrification, droplet-vitrification



CryoLetters 29(3), 243-251 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of Pelargonium apices by droplet-vitrification

Anthony Gallard1, Bart Panis2, Noëlle Dorion1, Rony Swennen2, and Agnès Grapin1*

1UMR GenHort (INRA, INH, UA), Institut National d’Horticulture, 49045 Angers Cedex 01, France
2 Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Department of Biosystems, K.U.Leuven, B3001 Leuven, Belgium
*Correspondence author: Agnès Grapin


The droplet-vitrification method was adapted to Pelargonium apices by optimizing the duration of the loading solution (LS) as well as the plant vitrification solution 2 (PVS2). The excised apices were dehydrated in two steps (20 min in LS and 15 min in PVS2) and then immersed directly in liquid nitrogen (LN). After thawing and unloading in the recovery solution at room temperature for 15 min, apices were plated onto semi-solid Murashige and Skoog medium. This simple protocol without any pretreatment was successfully applied to eight cultivars with a survival level ranging between 55.6 - 96.2% and a regrowth level between 9.1% - 70.6%. These results prove the feasibility of the long-term storage of Pelargonium germplasm through cryopreservation.

Keywords: LS, PVS2, P x hortorum, P x peltatum



CryoLetters 29(3), 253-260 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Nipawan Jitsopakul1, Kanchit Thammasiri2,3* and Keiko Ishikawa4

1Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
2Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
3Institute of Science and Technology for Research and Development, Mahidol University, Nakhonpathom 73170, Thailand.
4Department of Research and Development, Japan Horticultural Production and Research Institute, Chiba 270-2221, Japan.
*Correspondence author: Kanchit Thammasiri e-mail:


Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2% Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 ± 3°C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 ± 3°C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40°C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40% was observed with cryopreserved beads with 35% water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.

Keywords: Vanda coerulea, protocorms, cryopreservation, encapsulation-dehydration, morphological variation, ploidy level



CryoLetters 29(3), 261-268 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Simplified Technique of Human Ovarian Tissue Freezing: Quick Cooling from -36°C

V. Isachenko1,2*, E. Isachenko1,2, J. Reinsberg1, M. Montag1, J Weiss2, F. Braun1 and H. van der Ven1

1Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany
2Department of Obstetrics and Gynaecology, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany
*Corresponding author: V. Isachenko e-mail:


Standard protocol of freezing of human ovarian tissue presupposes the very slow cooling (-0.3°C/min) from auto-seeding to -40°C, then slow cooling (-10°C/min) to -140°C and then direct plunging into liquid nitrogen. The aim of this investigation was to compare the -10°C/min cooling rate of human ovarian tissue from -40°C to -140°C with the -220°C/min rate (direct plunging into liquid nitrogen) from -36°C. After post-thawing in vitro culture of tissue, hormonal activity as well as follicle viability was evaluated. After culture of fresh tissue pieces (Group 1), pieces after freezing/thawing with slow cooling (-10°C/min) from -40°C (Group 2) and pieces after freezing/thawing with direct plunging into liquid nitrogen (-220°C/min) from -36°C (Group 3), the supernatants showed estradiol 17-β concentrations of 481, 441 and 459 pg/ml, respectively (P>0.1), and progesterone concentrations of 9.05, 5.06, 4.87 ng/ml, respectively (P1-2,3 < 0.05; P2,3 > 0.1). It is concluded that 94, 96, and 98% follicles for Groups 1, 2 and 3, respectively (P > 0.1), were normal. Technique of human ovarian tissue cryopreservation with very slow cooling to -36 °C and then direct plunging into liquid nitrogen with -220°C/min cooling rate is tolerated without apparent detriment.

Keywords: human ovary, freezing, cooling rate, hormones, follicles



CryoLetters 29(3), 269 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

LETTER TO THE EDITOR: Cryoprotectants are defined by function, not chemistry

T.C. Hawes

Department of Zoology, University of Otago, Dunedin, P.O. Box 56, New Zealand



CryoLetters 29(3), 270 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Johannes Overgaard1 and Martin Holmstrup2

1Department of Zoophysiology, University of Aarhus, 8000 Aarhus C, Denmark
2National Environmental Research Institute, University of Aarhus, Department of Terrestrial Ecology, Vejlsø vej 25, 8600 Silkeborg, Denmark

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