Abstracts: CryoLetters 26 (4), 2005

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

CryoLetters 26 (4), 213-222 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Meiqin Liu, Xin Shen, Weilun Yin and Cunfu Lu*

College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China.


Ammopiptanthus mongolicus is the only evergreen broadleaf shrub endemic to the Alashan desert, northwest sand area of China, and can survive –30oC or an even lower temperature in winter. A modified solid-phase subtraction hybridization technique was developed to isolate and screen cDNAs whose transcripts increased in cold-treated A. mongolicus seedlings. Sequence analysis of the screened clones indicated that 11 clones had coding regions, with four of them containing a complete open reading frame. Nine of the 11 clones shared various degrees of homology with the genes found in the GenBank database and the other two were unidentified sequences. Sequence data further revealed that these accumulated transcripts encoded: three low molecular weight proteins (a late-embryogenesis protein and two cold acclimation responsive proteins); two photosynthesis-related proteins, (photosystem I subunit II precursor (PsaD) and photosystem II oxygen-evolving complex 33kDa subunit OEC33); a protease inhibitor; an adenosine triphosphatase and a 14-3-3 related protein. Analysis of the function of these proteins indicated that the low molecular weight proteins were associated with water holding ability of cytoplasm, photosynthesis-related proteins participated in the adjustments of photosynthetic apparatus to resist photoinhibition; 14-3-3 related protein could interact with adenosine triphosphatase to enhance ATPase activity and energy metabolism, and protease inhibitor is involved in the prevention of unwanted cell death caused by reactive oxygen species. We suggest that cold acclimation with low light intensity in A. mongolicus is a more complex interaction of low temperature, light, energy and signal than that assumed previously.

Keywords: Ammopiptanthus mongolicus, cold acclimation, subtraction hybridization



CryoLetters 26 (4), 223-230 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Paul Matejtschuk

Centre for Biological Reference Materials, National Institute for Biological Standards & Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire. EN6 3QG. UK.


Freeze drying plays a vital part in the preparation of many biological reference materials. The general approaches that may be adopted in the application of lyophilization to these biomolecules are discussed and the importance of analytical methodology and formulation optimization emphasised. Examples are given from a number of recently processed materials.

Keywords: Lyophilization, biological standards, thermal analysis, formulation



CryoLetters 26 (4), 231-238 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


J.G. Day1*, E.E. Benson2, K. Harding2, B. Knowles2, M. Idowu2, D. Bremner2, L. Santos3, F. Santos3, T. Friedl4, M. Lorenz4, A. Lukesova5, J. Elster6, J. Lukavsky6, M. Herdman7, R. Rippka7 and T. Hall8

1CCAP, Scottish Association for Marine Science, Dunbeg, Argyll, PA37 1QA, UK.
2University of Abertay Dundee, Bell St., Dundee, DD1 1HG, UK.
3Dept. Botânica, Univ.  Coimbra, 3000 Coimbra, Portugal.
4SAG, Albrecht-von-Haller-Inst. Pflanzenwissenschaften, Univ. Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany.
5Inst. Soil Biology CAS, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic.
6Inst.Botany CAS, Dukelska 135, CZ 379 83 Trebon, Czech Republic.
7Inst. Pasteur, 28 Rue de Dr Roux, 75724 Paris, France.
8AQUAARTIS, 19 rue de la Dutee, 44806 St. Herblain-cedex, France.


Microalgae are one of the most biologically important elements of worldwide ecology and could be the source of diverse new products and medicines. COBRA (The COnservation of a vital european scientific and Biotechnological Resource: microAlgae and cyanobacteria) is the acronym for a European Union, RTD Infrastructures project (Contract No.  QLRI-CT-2001-01645). This project is in the process of developing a European Biological Resource Centre based on existing algal culture collections. The COBRA project’s central aim is to apply cryopreservation methodologies to microalgae and cyanobacteria, organisms that, to date, have proved difficult to conserve using cryogenic methods. In addition, molecular and biochemical stability tests have been developed to ensure that the equivalent strains of microorganisms supplied by the culture collections give high quality and consistent performance. Fundamental and applied knowledge of stress physiology form an essential component of the project and this is being employed to assist the optimisation of methods for preserving a wide range of algal diversity. COBRA’s “Resource Centre” utilises Information Technologies (IT) and Knowledge Management practices to assist project coordination, management and information dissemination and facilitate the generation of new knowledge pertaining to algal conservation.  This review of the COBRA project will give a summary of current methodologies for cryopreservation of microalgae and procedures adopted within the COBRA project to enhance preservation techniques for this diverse group of organisms.

Key words: Algae, algal biotechnology, BRC, cryoinjury, cyopreservation, culture collection

This paper was originally presented at a joint meeting of the National Institute for Biological Standards and Control and the Society for Low Temperature Biology entitled “Current Trends in Cryobiology and Lyophilisation held 4-5th September 2003.



CryoLetters 26 (4), 239-250 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Theoretical analyses of thermal stress of blood vessel during cryopreservation

Gang Zhao1*, Zhi-Feng Liu1, Ai-Li Zhang2, Hai-Feng Zhang1, Shu-Xia Cheng1

1Department of Thermal Science and Energy Engineering, University of Science and Technology of China (USTC), Hefei 230027, P.R. China.
2 School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, P.R. China.
*To whom correspondence should be addressed; E-mail:


The infinite long hollow cylinder was selected as the model for studying the thermal stress inside the vascular wall during the common cryopreservation process (i.e. cooling-holding at a certain temperature-warming-holding at a second temperature). Transient distributions of temperature and stress were obtained; and effects of cooling rates, warming rates, the holding temperatures on this two distributions were analyzed. Coupled with the experimental results of other researchers, our theoretical predictions indicated that the appearance of the axial compressive stress during the initial period of cooling process was not the governing role that may cause the circumferential crack of the inner and outer vascular wall. Instead, it was the axial tensile stress during the initial period of the warming process that may cause the cracks. Furthermore, the stress history in the multi-steps warming methods was studied. Contrary to the common opinion, the results revealed that the multi-steps method could not decrease the maximal stress while it can change the temperature range containing the maximal stress from low temperature stage to relatively high temperature stage. As the flexibility of the blood vessel was partially recovered in the relative high temperature stage, it can bear the maximal stress that it cannot bear when still left in the low temperature stage, and the intrinsic feature of multi-steps warming method was revealed by this study. This paper provides a theoretical supplement to the study of Pegg D. E. et al (1997) and Buján J. et al (2001) published in the journal of Cryobiology.

Keywords: blood vessel, cryopreservation, thermal stress, crack



CryoLetters 26 (4), 251-258 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Mikio Fukuhara1* and Akiyoshi Kokuta2

1*Tungaloy Co.Ltd., 50,2-1, Kitakase, Saiwai, Kawasaki, 212-0057  JAPAN
2 Kohmix Co.Ltd., 51, 2-8, Tsuchidana-Naka, Ayase , 252-1113  JAPAN


The freezing point of pure water is expected to be about 163 K in the absence of hydrogen bonds for water molecules and no rotation of water molecules.  In anticipation of freezing point depression by breaking of the hydrogen bonds and formation of irrotational bonds with OH groups, a potassium-hydrated silicate solution was prepared. From thermal and ultrasonic analyses, we found that the silicate solution has a eutectic point of around 190 K in the SiO2-KOH-H2O system.  The differential scanning calorimetric (DSC) analysis showed a sharp exothermal peak of 45.7 J/g with the freezing point of 232 K. The FT-IR difference spectrum indicated that the solution is constructed of silanol structure with OH groups and siloxane linkage with (Si-O-Si) bonds.  The former is derived from hydration of water in the presence of potassium, while the latter makes the structure stable by being irrotationally bound for hydration shells, by analogy with the C-O bonds in protein with non-frozen water molecules.  The formation of the solution unfreezable down to 190 K might be due to both hydration and structural anchor effects.

Keywords: eutectic point of 190 K, potassium-hydrated silicate solution, irrotational bond, ATRIRFTS, DSC, ultrasonic analyses.



CryoLetters 26 (4), 259-688 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

cryopreservation of whole seeds and excised embryonic axes of citrus suhuiensis cv. Limau Langkat in accordance to their desiccation sensitivity

Makeen, A. Makeen1, Normah M. Noor1*, Stephane Dussert2 and Mahani M. Clyde3

1 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
2 IRD, 911 Avenue d’ Agropolis, BP 5045, F-34032 Montpellier Cedex 1. France.
3 School of Environmental Sciences and Natural Resources, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.


Following the investigation of desiccation sensitivity and freezing tolerance of the whole seed of Citrus suhuiensis cv. limau langkat, desiccation sensitivity and cryopreservation of the excised embryonic axes from the seeds of the same species were examined. Three drying conditions were employed: desiccation by equilibrium for the whole seeds and desiccation in laminar airflow and over silica gel for the excised embryonic axes. The relevance of desiccation sensitivity (WC50) to cryopreservation of whole seeds and excised axes was investigated. High desiccation tolerance (WC50 = 0.034 g H2O.g-1dw) was acquired for axes desiccated with faster dehydration rate (1.5 g.g-1.h-1) in laminar airflow compared to substantially lower desiccation tolerance (WC50 = 0.132 and 0.110 g H2O.g-1dw) acquired under slower dehydration rates (1.0 and 0.005 g.g-1.h-1) for axes desiccated over silica gel and whole seeds desiccated by equilibrium respectively. While few whole seeds (8.3%) survived freezing, high recovery percentages of axes (83.3% and 62.2%) after freezing were obtained under laminar airflow and silica gel drying conditions respectively. Irrespective of the drying method employed, axes survival percentages after exposure to LN temperature commensurate with the desiccation sensitivity pattern. For the whole seeds, a factor other than desiccation sensitivity that limits the tolerance to exposure to LN temperature seems to exist and still needs to be defined.

Keywords: Citrus suhuiensis cv. Limau langkat, cryopreservation, dehydration rate, desiccation sensitivity, embryonic axis.



CryoLetters 26 (4), 269-676 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Assessment of Desiccation Sensitivity of Tea Embryos for Cryopreservation

Haeng-Hoon Kim1*, Ju-Won Yoon1, Sang-Un Park2, Ju-Hee Kim3, Eun-Gi Cho1 and Florent Engelmann4, 5

1National Institute of Agricultural Biotechnology, Suwon 441-707, Korea. (email for H.H. Kim:
2Division of Plant Science and Resources, Chungnam National University, Daejeon 305-754, Korea.
3Bosung Tea Experiment Station, Bosung 546-804, Korea.
4Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address).
5International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


Tea (Camellia sinensis (L.) O. Kuntze) has been reported as a species with recalcitrant seeds. The seeds can be stored for less than one year under high humidity conditions in a refrigerator at 5-7°C. An efficient cryopreservation protocol for tea embryos using embryonic axes with portions of cotyledons still attached as drying material was established, which led to survival percentages around 92%. However, understanding the pattern of desiccation sensitivity, which is the key-limiting factor for cryopreservation, is of importance for implementation of cryopreservation using this protocol. In this study, the degree of desiccation sensitivity of tea seeds and cotyledonary embryonic axes (CEAs) was studied as a function of dehydration velocity, repeated dehydration-rehydration cycles, storage temperature, duration of storage of dried CEAs at room temperature, and seed harvesting date.

This study suggests that there are no less than two mechanisms involved in desiccation sensitivity of tea seeds and embryos. Firstly, desiccation sensitivity of tea embryos occurs predominantly in a quantitative manner with continuous variation under intermediate dehydrated status rather than because of desiccation itself to a critical moisture content (MC). Secondly, desiccation sensitivity is due to the removal of the structural water at MCs of lower than 11.5 %, when the EAs are flash-dried.

Keywords: Camellia sinensis (L.) O. Kuntze, cryopreservation, embryonic axes, desiccation sensitivity, rehydration, harvesting date.

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