Abstracts: CryoLetters 34 (2), 2013

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



Volume 34, No. 2 March/April 2013

ISSN 0143-2044



Cryopreservation for the ‘in perpetuity’ conservation of yam and cassava genetic resources
Dominique Dumet, Elohor Diebiru, Abigael Adeyemi,
Olubimpe Akinyemi, Badara Gueye, and Jorge Franco




Rapd and phytochemical analysis of Thymus moroderi plantlets after cryopreservation
Ana Marco-Medina and José Luis Casas




Genetic stability assessment of wasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis
Toshikazu Matsumoto, Takashi Akihiro, Shinya Maki,
Kouhei Mochida, Masaru Kitagawa, Daisuke Tanaka,
Shin-ichi Yamamoto and Takao Niino




The ice nucleation activity of extremophilic algae
Jana Kvíderová, Josef Hájek and Roger M. Worland




Production of yam mosaic virus (ymv)-free Dioscorea opposita plants by cryotherapy of shoot-tips
Jong Hee Shin, Dong Kyoon Kang and Jae Keun Sohn




“How important are internal temperature gradients in french straws during freezing of bovine sperm in nitrogen vapor?”
M.V. Santos, M. Sansinena, N. Zaritzky and J. Chirife




Effect of temperature and glycerol on the hydrogen-bond dynamics of water
Pavan K. GhattyVenkataKrishna and Edward C. Uberbacher




SLTB Abstracts Annual Meeting 2012, London
“Advances in Low Temperature Biology”






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CryoLetters 34 (2), 107-118 (2013)
© CryoLetters,

Cryopreservation for the ‘in perpetuity’ conservation of yam and cassava genetic resources

Dominique Dumet1*, Elohor Diebiru1, Abigael Adeyemi1,
Olubimpe Akinyemi1, Badara Gueye1, and Jorge Franco2

1Genetic Resources Center, IITA, Ibadan, Nigeria
2Biometrics Unit, IITA, Ibadan, Nigeria
*Corresponding author email:


Cryopreservation via droplet vitrification showed high efficiency for cassava meristems (79% average recovery) when these were excised from in vitro seedlings. The efficiency of the process dropped considerably (to >23%) when meristems were excised from field-grown plants, thus precluding the use of such explants for routine cryobanking. In yam, large disparities were observed in the ability of meristems to produce a shoot after cryopreservation ranging from 0 to 60%, depending on the accession. Overall, better recovery was observed for Dioscorea rotundata than for D. alata, the two main species tested. Using a probabilistic decision support tool and taking into consideration our cryoprocessing capacity, we conclude that processing 100 meristems per accession and retrieving 30 to estimate the recovery rate of the batch are a good compromise for the cryobanking routine.

Keywords: cryobanking, Dioscorea spp., Manihot esculenta, meristems, decision-support tool.

Abbreviations: DMSO, dimethyl sulfoxide; GLMM, Generalized Linear Mixed Model; MS, Murashige and Skoog; PVS2, plant vitrification solution 2




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CryoLetters 34 (2), 119-127 (2013)
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Ana Marco-Medina and José Luis Casas*

Laboratorio de Biotecnología Vegetal, Instituto Universitario de Investigación CIBIO (Centro Iberoamericano de la Biodiversidad), Universidad de Alicante, Crta. San Vicente del Raspeig s/n. E-03690 San Vicente del Raspeig, Alicante (Spain).
*Corresponding author  e-mail:


Cryopreservation is at present the most reliable strategy to preserve plant germplasm. When aromatic plants are the object of conservation it is necessary to assess not only the genetic but also the phytochemical stability to ensure that plant material maintains its qualities after storage. In this work we present molecular and phytochemical stability data related to a previously described vitrification-based cryopreservation protocol for Thymus moroderi Pau ex Martínez. RAPD markers have been used to assess the genetic stability of T. moroderi explants and revealed 0.34% of variation in the cryopreserved material studied. Phytochemical data collected from GC-MS analysis of dichloromethane extracts from cryopreserved plantlets rendered a profile in which 1,8-cineole (14.5%), camphor (5.9%) and borneol (5.2%) were the major components. Both data confirmed the suitability of the cryopreservation protocol applied.

Keywords: cryopreservation, essential oils, molecular marker, RAPD, thyme, vitrification.




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CryoLetters 34 (2), 128-136 (2013)
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*Toshikazu Matsumoto1, Takashi Akihiro1, Shinya Maki2,
Kouhei Mochida3, Masaru Kitagawa3, Daisuke Tanaka4,
 Shin-ichi Yamamoto4 and Takao Niino4

1Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-1060, Japan.
2Niihama National College of Technology, Niihama, Ehime 792-8580, Japan.
3Shimane Agricultural Technology Center, Izumo, Shimane 693-0035, Japan.
4National Institute of Agrobiological Sciences (NIAS), Tsukuba, Ibaraki 305-8518, Japan.
*Corresponding author  e-mail:


This study compared the effect of cryopreserved storage duration of wasabi shoot tips, which derived from the same in vitro mother-plant. We compared the survival of shoot tips and the genetic stability of regenerated plants originating from four experimental groups: shoot tips stored in a -150°C deep-freezer for 10 years; shoot tips stored in liquid nitrogen for 2 h; shoot tips treated with PVS2 vitrification solution; and untreated controls. No significant difference in survival was observed between the four experimental groups. Survival ranged between 93 and 100%. Genetic stability of plants regenerated from cryopreserved shoot tips was assessed over a period of 24 months using morphological, biochemical and molecular markers. While glucose, fructose and glutamic acid concentrations differed slightly between experimental groups after 16 months, these differences disappeared after 24 months. No significant differences were noted for the morphological markers studied (petiole length, shoot number and leaf index). No differences were observed in RAPD profiles obtained with the six primers tested.

Keywords: wasabi, genetic stability, morphological, biochemical, molecular




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CryoLetters 34 (2), 137-148 (2013)
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The ice nucleation Activity of extremophilic alga

Jana Kvíderová1*, Josef Hájek2 and Roger M. Worland3

1Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, CZ-379 82 Třeboň, Czech Republic (
2Laboratory of Photosynthetic Processes, Department of Plant Physiology, Institute of Experimental Biology, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic (
3British Antarctic Survey, High Cross, Madingley Road, Cambridge, CB3 0ET, United Kingdom (
*Corresponding author  email:


Differences in the level of cold acclimation and cryoprotection estimated as ice nucleation activity in snow algae (Chlamydomonas cf. nivalis and Chloromonas nivalis), lichen symbiotic algae (Trebouxia asymmetrica, Trebouxia erici and Trebouxia glomerata), and a mesophilic strain (Chlamydomonas reinhardti) were evaluated. Ice nucleation activity was measured using the freezing droplet method. Measurements were performed using suspensions of cells of A750 (absorbance at 750 nm) ~ 1, 0.1, 0.01 and 0.001 dilutions for each strain. The algae had lower ice nucleation activity, with the exception of Chloromonas nivalis contaminated by bacteria. The supercooling points of the snow algae were higher than those of lichen photobionts. The supercooling points of both, mesophilic and snow Chlamydomonas strains were similar. The lower freezing temperatures of the lichen algae may reflect either the more extreme and more variable environmental conditions of the original localities or the different cellular structure of the strains examined.

Keywords: ice nucleation, lichen symbiotic algae, snow algae, supercooling point




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CryoLetters 34 (2), 149-157 (2013)
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Production of YAm mosaic virus (YMV)-free Dioscorea opposita pLantS by cryotherapy OF SHOOT-TIPS

Jong Hee Shin1*, Dong Kyoon Kang1 and Jae Keun Sohn2

1Gyeongbuk Provincial Agricultural Technology Administration, Daegu, Republic of Korea, (
2Department of Agronomy, Kyungpook National University, Daegu, Republic of Korea (
*Corresponding author  email:


In the present study, Yam mosaic virus (YMV) could be efficiently eliminated by cryotherapy in Dioscorea opposita. Shoot apices were precultured for 16 h with 0.3 M sucrose, encapsulated in sodium alginate and dehydrated for 4 h prior to direct immersion in liquid nitrogen. Up to 90% of the plants regenerated from cryopreserved shoot tips were YMV-free, whereas only 40% of those regenerated using meristem culture were YMV-free. YMV-free yam plantlets could be propagated in vitro through nodal stem culture, with sequential subculturing at 6-week intervals on medium containing 0.5 mg l-1 kinetin. The microtubers formed at the bottom and axil of the explants, incubated at 30°C after being chilled (4°C) for 3 months, could be sprouted successfully under in vivo conditions. Healthy plants were established without any damaging symptoms of the virus. Thus, cryotherapy provides an alternative method for efficient elimination of yam viruses, and could be simultaneously used for long-term storage of yam germplasm and for the production of virus-free plants.

Keywords: Cryotherapy, yam mosaic virus (YMV), yam, in vitro propagation, virus-free.




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CryoLetters 34 (2), 158-165 (2013)
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“How important are internal temperature gradients in French straws during freezing of bovine sperm in nitrogen vapor?”

M.V. Santos1, M. Sansinena2*, N. Zaritzky1 and J. Chirife2

1Depto. de Ingeniería Química, Facultad de Ingeniería, Universidad Nacional de La Plata and Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CONICET-UNLP) Calle 47 y 116, La Plata 1900, Argentina
2Facultad de Ciencias Agrarias, Pontificia Universidad Católica Argentina, Cap. Gral. Ramón Freire 183, 1426 Buenos Aires, Argentina.
*Corresponding author  email:


The subject of present work was to predict internal temperature gradients developed during freezing of bovine sperm diluted in extender, packaged in 0.5 ml French plastic straws and suspended in static liquid nitrogen vapor at -100ºC. For this purpose, a mathematical heat transfer model previously developed to predict freezing times (phase change was considered) of semen/extender packaged in straw was extended to predict internal temperature gradients during the cooling/freezing process. Results showed maximum temperature differences between the centre and the periphery of semen/extender “liquid” column was 1.5ºC for an external heat transfer coefficient, h = 15 W/(m2 K), and only 0.5ºC for h = 5 W/(m2 K). It is concluded that if a thermocouple wire were inserted in a 0.5 ml plastic straw to monitor the freezing process in nitrogen vapor, its radial position would have little importance since expected internal gradients may be safely neglected. This finding facilitates the interpretation of freezing rates in 0.5 ml plastic straws immersed in nitrogen vapor over liquid nitrogen, a widely used method for cryopreservation of bovine spermatozoa.

Keywords: Freezing, bovine sperm, heat transfer coefficient, temperature gradients, nitrogen vapor, finite element method, French plastic straws.




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CryoLetters 34 (2), 166-173 (2013)
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Effect of temperature and glycerol on the hydrogen-bond dynamics of water

Pavan K. GhattyVenkataKrishna and Edward C. Uberbacher

Computational Biology and Bioinformatics Group, Oak Ridge National Laboratory, Oak Ridge, TN 37830
Corresponding author email:


The effect of glycerol, water and glycerol-water binary mixtures on the structure and dynamics of biomolecules has been well studied. However, a lot remains to be learned about the effect of varying glycerol concentration and temperature on the dynamics of water. We have studied the effect of concentration and temperature on the hydrogen bonded network formed by water molecules. A strong correlation between the relaxation time of the network and average number of hydrogen bonds per water molecules was found. The radial distribution function of water oxygen and hydrogen atoms clarifies the effect of concentration on the structure and clustering of water.

Keywords: hydrogen bonds, relaxation time, glycerol-water, bio-protective solvents

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