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Abstracts: CryoLetters 23 (4), 2002

CryoLetters 23, 209-216 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of tea (Camellia sinensis L.) seeds and embryonic axes

Haeng-Hoon Kim1*, Young-Soon Cha1, Hyung-Jin Baek1, Eun-Gi Cho1, Young-Am Chae2 and Florent Engelmann3, 4

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea. (*hkim@rda.go.kr).
2Department of Agronomy and Research Center for New Biomaterials in Agriculture, Seoul National University, Suwon 441-744, Korea.
3International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
4Institut de recherche pour le développement (IRD), BP 64501, 34394 Montpellier Cedex 05, France. (current address).

Abstract

This study investigated the tolerance to desiccation and freezing of tea seeds, embryonic axes (EAs) and cotyledonary embryonic axes (CEAs, consisting of EAs with portions of cotyledons still attached). No seeds germinated after desiccation and cryopreservation. EAs extracted from seeds desiccated to 18.9% moisture content (fresh weight basis) and cryopreserved showed 20.7% survival but plantlet production from these EAs was impossible. When EAs and CEAs were extracted from seeds before being submitted to desiccation and freezing, survival of control and frozen samples was equivalent with both types of materials. However, plantlet production was significantly higher from control and cryopreserved CEAs than EAs. The maturity stage of the seeds from which CEAs were extracted had an important effect on their survival and plant production percentages, mature seeds providing better results than early mature and late mature seeds. The highest percentages of plantlet production from cryopreserved CEAs, which ranged between 75.1 and 80.4%, were achieved for EA moisture contents between 21.5 and 15.0%.

Keywords: Camellia sinensis L.; desiccation; cryopreservation; embryonic axes; cotyledonary embryonic axes.

 

 

CryoLetters 23, 217-228 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Survival at low temperatures in insects: What is the ECOLOGICAL significance of THE SUPERCOOLING POINT?

D. Renault1*, C. Salin1, G. Vannier2 and P. Vernon1

1Station biologique de Paimpont, UMR 6553 CNRS, Université de Rennes-I, 35380 Paimpont, France. Tel: +33 (0)2.99.61.81.69 : Fax: +33 (0)2.99.61.81.87 e-mail: david.renault@univ-rennes1.fr
2Laboratoire d'écologie générale, UMR 8571 CNRS, Muséum national d'histoire naturelle, 4, avenue du Petit-Château, 91800 Brunoy, France

Abstract

Many freezing-intolerant insects may die during long or even brief exposures to temperatures above their supercooling point (SCP). Consequently, the real ecological value of the SCP remains ambiguous, particularly for tropical species that never experienced cold exposures. The bimodal distribution of SCP is discussed in the light of sexual dimorphism. The importance of sex in insect cold hardiness has been regularly neglected and although we admit that in some species sex may be uneasy to determine, it should be taken into account in further studies. We suggest that supercooling ability may be, at least partially, a result of adaptations to other functions unrelated to cold, including the desiccation resistance. The potential causes of insect death at low temperatures during survival experiments have also been examined. Prolonged exposures at lethal low temperatures can produce deleterious effects (including death) even if the insect does not freeze; during long-term exposure to low temperatures the organisms may finally die from the exhaustion of energy reserves.

Keywords: survival, supercooling points, low temperatures, chilling injury, starvation, sexual dimorphism, tropical species.

 

 

CryoLetters 23, 229-236 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A MODIFIED DIFFERENTIAL SCANNING CALORIMETRY FOR DETERMINATION OF CELL VOLUMETRIC CHANGE DURING THE FREEZING PROCESS

Dawei Luo1,2, Xu Han1, Liqun He2, Xiangdong Cui 1, Shuxia Cheng2, Caicheng Lu3, Jianghan Liu4 and Dayong Gao*1,2

1Department of Mechanical Engineering and Center for Biomedical Engineering, University of Kentucky, Lexington, KY, U.S.A. 40506.
2Cryobiology Research Institute and Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei, Anhui, China.
3Department of Electrical Engineering, University of Kentucky, Lexington, KY, U.S.A.
4Department of Blood Transfusion, 301 Hospital, Beijing, China.

 Abstract

A modified analytical and experimental method using differential scanning calorimeter (DSC) was developed to determine the cell volume change during the freezing process. Two cell types were used in the study: human platelets and erythrocytes (red blood cells). Isotonic cell suspensions with different cytocrits were prepared and used in the DSC experiments. Low cooling rates were used to avoid intracellular ice formation. Cell suspensions were cooled from room temperature to –40C. Latent heat release from the freezing of cell suspensions was shown to be a linear function of cytocrit. From slope and intercept of the linear function, cell volume change was determined based on a developed theoretical model. From experimental data and theoretical analyses, it was revealed that (a) the final volume of a human platelet at –40C was 33.7% of its isotonic volume, and 15.2% of the original (at isotonic condition) intracellular water remained unfrozen inside platelets, and (b) the final volume of human erythrocyte at –40C was 50.0% of its isotonic volume, and 30.3% of the original intracellular water was kept inside cells as residual unfrozen water.

Keywords: cryopreservation, cell volume change, DSC, human platelets, human red blood cells/erythrocytes.

 

 

CryoLetters 23, 237-244 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane (Saccharum sp.) embryogenic calluses

M.E. Martínez-Montero1*, N. Mora1, J. Quiñones1, M.T. González-Arnao2, F. Engelmann3 and J.C. Lorenzo1

1Laboratorio de Mejoramiento Genético, Centro de Bioplantas, Universidad de Ciego de Avila, CP 69450, Ciego de Avila, Cuba. * For correspondence (Email for M.E.M-M: cubamarcose00@hotmail.com)
2Facultad de Biología, Universidad de La Habana, Calle 25 e/ J e I, Vedado, La Habana, Cuba.
3Institut de Recherche pour le Développement, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex 05, France.

Abstract

In this paper, we investigated if the differences consistently noted in survival and plantlet production between cryopreserved and non-cryopreserved, control sugarcane embryogenic calluses were related to modifications induced during cryopreservation in the structural and functional integrity of cell membranes. For this, the evolution of electrolyte leakage, lipid peroxidation products and cell membrane protein contents was measured during 5 d after cryopreservation. Differences between control and frozen calluses were observed only during the first 2 (electrolyte leakage) or 3 d (lipid peroxidation products and membrane protein content) after freezing. It was not possible to link these differences with the differences noted in survival and plant production between control and cryopreserved calluses. Additional studies are thus needed to elucidate which biochemical factors, linked to survival and plantlet regeneration, are affected by cryopreservation.

Keywords: Saccharum sp., electrolyte leakage, cell membrane proteins, malondialdehyde, aldehydes, lipid peroxidation.

 

 

CryoLetters 23, 245-254 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

AMMONIUM DETOXIFYING ACTIVITY IS MAINTAINED AFTER 72 HOURS of COLD PRESERVATION OF RAT HEPATOCYTES IN UNIVERSITY OF WISCONSIN (uw) SOLUTION

Sebastián D. Calligaris1, Luciana L. Almada1, Edgardo E. Guibert2, Claudio Tiribelli3 and Joaquin V. Rodriguez1*

1 Farmacología, Departamento de Ciencias Fisiológicas, and
2 Biología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531- 2000 Rosario (S2002LRK), Argentina.
3Centro Studi Fegato, Department of Biochemistry, Biophysics and Chemistry of Macromolecules (BBCM), University of Trieste, Via Giorgieri 1, 34127 Trieste, Italy.
* E-mail:
jcravero@infovia.com.ar

Abstract

The ammonium removal efficiency (ARE) and cell viability was investigated in freshly isolated rat hepatocytes exposed to increasing ammonium loads (0.1-2.0 mM). No difference was observed in both ARE and cell viability at the different ammonium concentrations tested. Storage of hepatocytes at 0 °C for 72 hours inhibited ammonium removal and urea synthesis. Rewarming of cells at 37 º C for 120 min was followed by an ARE fully comparable to freshly isolated hepatocytes. These data indicated that cold preservation of rat hepatocytes for 3 days in UW followed by a rewarming is associated with normal ammonium detoxification efficiency

Keywords: ammonium, hepatocytes, UW, detoxification, cold preservation

 

 

CryoLetters 23, 255-262 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

SEMEN CRYOPRESERVATION: A GENETIC EXPLANATION FOR SPECIES AND INDIVIDUAL VARIATION?

Lisa M. Thurston*1, Paul F. Watson1 & William V. Holt2

1Veterinary Basic Sciences, Royal Veterinary College Royal College Street, London, NW1 0TU, UK.
2Institute of Zoology, Zoological Society of London, Regents Park, London, NW1 4RY, UK.

Abstract

Although semen cryopreservation has been applied successfully in a few species, considerable variation in post-thaw semen viability exists. Independent of sperm quality before freezing, the semen of certain individuals will consistently freeze badly, resulting in poor motility, disrupted acrosome and plasma membrane, and thus reduced fertilising ability, indicating the existence of variation in membrane properties within species. A more comprehensive understanding of sperm cryobiology would be obtained by the investigation of within-species variation in the susceptibility of spermatozoa to cryoinjury. This review aims to explore the phenomenon of consistent variation in frozen semen quality between species and between individuals in an effort to find new insights into the reasons for cryoinjury. Recent studies suggest that there is a genetic basis for variation in post-thaw semen quality, and argue that modern molecular technologies are able to identify markers linked to genes influencing this variation. The identification of genetic differences between individuals, which may be linked to cryosurvival, provides an opportunity to develop a functional and molecular understanding of the factors that influence semen cryopreservation, allowing selective breeding of desired traits and the development of genetic tests that predict the outcome of semen freezing.

Keywords: AFLP, genetics, freezing, cryopreservation, morphology, sub-population, variation

This paper is a contribution to a symposium of the Society for Low Temperature Biology in October 2002.

 

 

CryoLetters 23, 263-268 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

CHONDROCYTE RECOVERY IN CRYOPRESERVED PORCINE ARTICULAR CARTILAGE AFTER BONE CARRIER ALTERATION

N.M. Jomha*, P.C. Anoop and L.E. McGann

University of Alberta, Edmonton, Alberta, Canada.
* correspondence address:  Nadr M Jomha, MD, MSc, FRCS(C), 2D2.32 WCMC, University of Alberta Hospital, 8440-112 St, Edmonton, Alberta, Canada, T6G 2B7. Tel: 780 407 2806. Fax 780 407 6505. Email:
nadr@telusplanet.net

Abstract

In order to investigate the consequences on the distribution of cell recovery through a cross-section of articular cartilage, the pathway for ice nucleation and diffusion of water and solutes in porcine osteochondral tissue was altered by drilling a 2mm diameter hole through the subchondral bone to the base of the cartilage. Samples equilibrated with 1M dimethyl sulfoxide were cooled at 1°C/min to -30°C then stored in liquid nitrogen. A significant increase in chondrocyte recovery was documented when compared to samples cryopreserved without holes (48.3%:28.6%, P=0.003). The most significant change due to bone base modification was an increase in recovery in the middle section of the cartilage. These results provide insight into mechanisms of cryoinjury in tissue systems

Keywords: cryopreservation, articular cartilage, porcine, chondrocyte recovery

 

 

CryoLetters 23, 269-274 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECTS OF A FREEZING EVENT DURING HIBERNATION ON FURTHER SURVIVAL, REPRODUCTION AND GROWTH IN THE PARTIALLY FREEZING TOLERANT LAND SNAIL Helix aspersa MÜLLER (GASTROPODA: HELICIDAE)

Armelle Ansart1*, Philippe Vernon2 and Jacques Daguzan1

1Université de Rennes 1, UMR 6553 Ecobio, bât. 14, 263 Av. Gal Leclerc, CS 74205, 35042 Rennes Cedex, France, email: armelle.ansart@univ-rennes1.fr
2Université de Rennes 1, UMR 6553 Ecobio, Station Biologique, 35380 Paimpont, France

Abstract

Tolerance of ectothermic animals to freezing is often estimated by assessing survival a few days after the treatment. However, in the long term, ice formation in the body tissues can affect survival, as well as reproductive capability and growth. The land snail Helix aspersa survives only short durations with ice in its tissues, to a lethal limit of 40 to 60 % of its body water frozen. Adult and immature snails were treated during their winter dormancy period to a freezing event above this limit; their survival was observed both in the short and long term, as well as their ability to reproduce (adults) and grow (immature snails). Treated snails were compared with a control group, which was not frozen. No difference appeared in the survival, reproduction and growth of control and frozen snails. This study confirms partial freezing tolerance in this population of Helix aspersa.

Keywords: Cold hardiness, Freezing tolerance, Survival, Growth, Reproduction, Helix aspersa

 

 

CryoLetters 23, 275-276 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

Letter to the editor -
"ICE STRUCTURING PROTEINS" – SUBSTANCE OR SEMANTICS

Felix Franks, London, UK

 

 

CryoLetters 23, 277-278 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

Letter to the editor -
IS APOPTOSIS AN IMPORTANT MECHANISM OF CRYOINJURY  IN VIVO?

J.C. Bischof, N.E Hoffmann, and K.R Roberts, Minneapolis, Minnesota, USA

J.E Coad, Morgantown, West Virginia, USA

 

 

CryoLetters 23, 279-280 (2002)
© Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

Letter to the editor -
IS APOPTOSIS AN IMPORTANT MECHANISM OF CRYOINJURY  IN VIVO? – A REPLY

Andrew Gage, Webster Wisconsin USA and John Baust, Binghamton, New York, USA

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