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Abstracts: CryoLetters 26 (6), 2005

CryoLetters 26 (6), 341-348 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF PRUNUS AVIUM L. EMBRYOGENIC TISSUES

Ghislaine Grenier-de March*1, Marie-Thrèse de Boucaud 2 and Paweł Chmielarz 3

1Institut Supérieur d'Agriculture de Beauvais (ISAB), Laboratoire de Biotechnologies Végétales, Rue Pierre Waguet - BP 30313, 60026 Beauvais Cedex, France
2Universit de Bordeaux I, Laboratoire de Physiologie Cellulaire Vgtale, Avenue des Facults, Bordeaux, 33405 Talence, France
3Polish Academy of Sciences (PAS), Institute of Dendrology, 62-035 Kórnik, Parkowa 5, Poland
*Corresponding author, e-mail:
ghislaine.grenier@isab.fr

Abstract

Embryogenic tissues from wild cherry (Prunus avium L.) were successfully cryopreserved by using a one-step freezing procedure. Cryoprotection consisted of a pretreatment on solid medium with increasing sucrose concentrations (0.25 M for 1 day, 0.5 M for 1 day, 0.75 M for 2 days, and 1.0 M for 3 days), followed by air desiccation to about 20% moisture content (fresh weight basis). This method was compared with a pretreatment on solid medium containing 5% DMSO and 2% proline, followed by immersion in a modified PVS2 cryoprotective solution. Pretreatment on solid medium with increasing concentrations of sucrose led to regrowth of frozen embryogenic tissues, and after 6 weeks of culture, growth was comparable to that of non-dehydrated and non-frozen tissues. By contrast, no regrowth was observed when embryogenic tissues were submitted to the solid/liquid pretreatment with DMSO/proline and a  modified PVS2 solution.

Keywords: cryopreservation, desiccation, rapid cooling, somatic embryos, sucrose, wild cherry

 

 

CryoLetters 26 (6), 349-356 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF QUERCUS ROBUR L. EMBRYOGENIC CALLI

Paweł Chmielarz1*, Ghislaine Grenier-de March2 and Marie-Thrèse de Boucaud3

1Polish Academy of Sciences, Institute of Dendrology, 62-035 Kórnik, Parkowa 5, Poland.
2Institut Supérieur d'Agriculture de Beauvais (ISAB), Laboratoire de Biotechnologies Végétales, Rue Pierre Waguet - BP 30313, 60026 Beauvais Cedex, France.
3Universit de Bordeaux I, Laboratoire de Physiologie Cellulaire Vgtale, Avenue des Facults, Bordeaux, 33405 Talence, France.
*Corresponding author, e-mail:
pach@rose.man.poznan.pl

Abstract

Embryogenic calli of pedunculate oak (Quercus robur L.) were cryopreserved using direct immersion in liquid nitrogen. The pretreatment consisted of culture on a solid medium with increasing sucrose concentrations (0.25 M for 1 day, 0.5 M for 1 day, 0.75 M for 2 days, and 1.0 M for 3 days), followed by air desiccation of embryogenic calli to 17.3% (fresh weight basis). This method of cryoprotection was compared to a liquid cryoprotection treatment using high concentrations of sucrose solutions, followed by glycerol solutions. Regrowth of frozen tissue pretreated on a solid medium was significantly higher than those pretreated in the liquid solutions (Fig. 2).

Keywords: embryogenic calli, somatic embryos, cryopreservation, desiccation, sucrose

 

 

CryoLetters 26 (6), 357-366 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

IMPROVEMENT OF CRYOPRESERVATION RESULTS IN GARLIC USING LOW TEMPERATURE PRECULTURE AND HIGH-QUALITY IN VITRO PLANTLETS

E. R. Joachim Keller

Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany.
E-mail:
keller@ipk-gatersleben.de

Abstract

The efficiency of garlic cryopreservation is, amongst other factors, depending on the origin of the donor explants. So far, in vitro grown material has always been the least responding one with respect of the regrowth rates. On the other side, the possibility to produce virus-free material via meristem culture and to keep these clones then under isolated conditions in a clean culture induced studies to increase the efficiency of cryopreservation using this kind of material. Experiments have been performed to use various materials and cultivation temperatures for a vitrification protocol. Best results (up to 70 % regrowth) were obtained with cultures grown for only 10 months under in vitro conditions including a cold preculture of two months either at alternating or at permanently low temperatures. The conclusion was drawn that the quality of the explants and temperature conditions play a major role for the efficiency of cryopreservation using in vitro plantlets.

Keywords: garlic, cryopreservation, vitrification, PVS3, low-temperature preculture, in vitro storage

 

 

CryoLetters 26 (6), 367-378(2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ULTRASTRUCTURE OF Gentiana tibetica PROEMBRYOGENIC CELLS BEFORE AND AFTER COOLING TREATMENTS

Anna Mikuła1*, Teresa Tykarska2 and Mieczysław Kuraś2

1Botanical Garden - Center for Biological Diversity Conservation, Polish Academy of Sciences, ul. Prawdziwka 2, 02-973 Warsaw, Poland.
*e-mail:
amikula@ob.neostrada.pl
2Department of Plant Morphogenesis, Faculty of Biology, Warsaw University, ul. Miecznikowa 1, 02096 Warsaw, Poland

Abstract

The influence of increased concentrations of sucrose, 0.4 M sorbitol, DMSO and vitrification solution (PVS2) on the ultrastructure of non-frozen and frozen suspensions of Gentiana tibetica King ex Hook. F.tissue cells was investigated. Embryogenic aggregates were composed of three groups of cells of different size with various types of plastids. The ultrastructural changes resulting from increasing the sucrose concentration in the medium from 3 % to 6 % for 4 weeks and from treatment with 0.4 M sorbitol for 48 h were similar. Observations showed replacement of large vacuoles by numerous small ones, condensation of cytoplasm, accumulation of starch, and fragmentation of endoplasmic reticulum. Treatment with PVS2 led to degradation of starch, coalescence of amyloplasts and to shrinking of nucleoli from the third group of cells when originating from 6 % sucrose medium. The mitochondria initially had various shapes, but after PVS2 treatment showed only spherical shapes with sparse cristae. After programmed freezing of tissue protected by sorbitol and DMSO, lethal damage was observed: membrane and nuclei degradation, and cell destruction. Reversible changes after freezing were observed in tissue pretreated with vitrification solution: dilation of cell membranes, mitochondria with electron-lucent vessels, aggregation of numerous vesicles, and degradation of starch in amyloplasts. In cells cooled by a vitrification method, cell organelles appeared normal as early as 5 h after thawing, and anomalies were not observed after 48 h of post-thawing culture.

Keywords: Tibetan gentian, suspension culture, programmed freezing, vitrification, microscopic and ultrastructural analysis.

 

 

CryoLetters 26 (6), 379-386 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

COLD PRESERVATION OF ENDOTHELIAL CELLS IN SUCROSE-BASED SOLUTION (SBS) AND UNIVERSITY OF WISCONSIN (UW) SOLUTIONS: COMPARISON OF NORMOXIC OR HYPOXIC STORAGE

M.Hawkins, K.M.Sales, S.Dijk, B.Fuller*

University Department of Surgery, Royal Free Hospital & University College Medical School, London NW3 2QG, UK

Abstract

Cold preservation of endothelial cells was studied, comparing primary endothelial cells (human umbilical vein endothelial cells – HUVECs) and a continuously growing cell line (ECV304 cells). Viability at the end of 24h cold preservation was measured by dye exclusion, whilst metabolism was assessed by Alamar blue conversion. Two preservation solutions were studied (UW solution) and sucrose-based (SbS) in both cell types. The response was similar in both cell types to preservation under normoxic conditions (with percentage dye exclusion maintained at about 80% in both preservation solutions) whereas under hypoxic conditions ECV304 were more sensitive to preservation in UW solution (dye exclusion reduced to 43.5 ± 1.4% versus 73.6 ± 14% (P<0.01). Metabolism assessed by Alamar blue conversion after cold preservation and rewarming was similar in both ECV304 and HUVECs after storage under normoxic conditions in UW solution, but in both cell types, metabolism was higher in SbS (P<0.05 and p<0.01) than in UW solution. Under hypoxic conditions, both cell types showed similar recovery of metabolism after storage in either UW or SbS. If the cells (in this case ECV304 under aerobic conditions) were stored for 24h and then allowed to rewarm in either of the respective preservation solutions (UW or SbS for 1h) before the Alamar blue test, metabolism was higher (p<0.01) in those exposed to SbS. UW solution and SbS provide similar protection for endothelial cells under hypoxic conditions, but SbS has some advantages under normoxic storage or if the cells experience variable temperatures in the presence of residual preservation solution at the end of cold preservation period.

Keywords: Endothelial cells, cold preservation, UW solution, sucrose-based solution, Alamar blue assay.

 

 

CryoLetters 26 (6), 387-394 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECT OF CRYOHYPOBIOSIS ON CONSERVATION OF HUMAN BLOOD LEUKOCYTES

E.P. Svedentsov, E.S. Stepanova, T.V. Tumanova, O.O. Shcheglova, O.N. Devetyarova and S.V. Utyomov*

Blood Cryophysiology Laboratory (E.P. Svedentsov) of the Physiology Institute at the Scientific Center affiliated to the Ural branch of the Russian Academy of Sciences, Komi Republic, Syktyvkar, Russia.
*Kirov Research Institute of Hematology and Blood Transfusion, Kirov, Russia
Address for correspondence: Evgeni Pavlovich Svedentsov, apt. 28, 109 Karl Liebknect str., Kirov 610017, Russia. Tel. (8332) 37-26-22 (Office).

Abstract

Morphological and functional indicators were investigated of donor leukocyte concentrates mixed at a ratio 1:1 with three versions of cold-protective and kept at an undercooling temperature (-10ºC) for 24 hours, and with a cryoprotector of optimal composition for 12 days,. An optimal version of a non-freezing cold-protective solution was found. It was determined that 76 ± 4 % of the neutrophils retain there ability of phagocytosis among the viable cells (81 ± 5 %) after a 12 day hypobiosis. Thus the results testify to a high morphofunctional safety of human blood leukocytes which experienced cold-hypobiosis        (-10ºC) in an original non-freezing solution not requiring washing after re-warming.

Key words : leukocytes, neutrophils, cryohypobiosis, cryoprotector.

 

 

CryoLetters 26 (6), 395-399 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

SOME ATTRIBUTES OF COLD HARDINESS OF THE GREGARIOUS ECTOPARASITOID COLPOCLYPEUS FLORUS (HYMENOPTERA: EULOPHIDAE)

Panagiotis G. Milonas1* and M. Savopoulou-Soultani2

1 Benaki Phypathological Institute, Department of Entomology and Agricultural Zoology, Biological Control lab., 8 S. Delta str., 145 61 Kifissia, Greece.
Email:
biocon@bpi.gr
2 Laboratory of Applied Zoology and Parasitology, Faculty of Geotechnical Sciences Department of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

Abstract

Cold hardiness of diapause and non-diapause larvae of the parasitoid Colpoclypeus florus Walker (Hymenoptera: Eulophidae) was examined in the laboratory.  Mean supercooling point (SCP) for diapausing larvae was -26.7C and for non-diapausing larvae immediately after their larval development, was -16.2C. Mean SCP for non-diapause larvae at the prepupa stage was –19.1C. A short period of acclimation (1 week at 5C) had no influence on the mean SCPs of both diapause and non-diapause larvae. Pre-freeze mortality for diapause and non-diapause larvae was also studied. A constant exposure of diapause larvae to -6C resulted in high mortality (70.7%) after a period of 40 days.  In contrast, 6 days at -6C were sufficient to cause the same level of mortality in non-diapause larvae.  After exposure of 15 days at -9C, mortality for non-diapause larvae was 70%, whereas after 20 days at the same temperature mortality of diapause larvae was 25%.  The importance of these findings for the cryobiology of C. florus is discussed.

Keywords: Diapause, supercooling point, freeze intolerance, acclimation, pre-freeze mortality.

 

 

CryoLetters 26 (6), 402-408 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

PROBABILITY OF LETHAL DAMAGES OF CRYOPRESERVED BIOLOGICAL OBJECTS DURING STORAGE

Vysekantsev I.P., Gurina T.M., Martsenyuk V.F., Petrenko T.F., Kudokotseva E.V., Koshchiy S.V., Groshevoy M.I.

Institute for problems of cryobiology & cryomedicine of the National Academy of Sciences of the Ukraine, 23 Pereyaslavskaya str., 61015, Kharkov, Ukraine,
e-mail:
cryo@online.kharkov.ua

Abstract

This study demonstrated that with a cyclic change of the temperature from –196 up to –130 or –100C part of cryopreserved microorganisms, tumor and human embryo cells died. This should be taken into account when moving and transporting the cryopreserved objects. Storage of samples under the above-mentioned stable temperatures did not result in additional death of cells.

Keywords: cryopreservation, temperature gradients, bacteria, yeast, tumor cells, embryonic cells.

 

 

CryoLetters 26 (6), 409-411 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

LETTER TO THE EDITOR: ANALYSIS OF THERMO-MECHANICAL STRESS IN CRYOPRESERVATION

Yoed Rabin (rabin@cmu.edu) and Paul S. Steif (steif@cmu.edu)

Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA

 

 

CryoLetters 26 (6), 412 (2005)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

LETTER TO THE EDITOR: A RESPONSE

Zhao Gang

Department of Thermal Science & Energy Engineering, University of Science and Technology of China, HeFei 230027, P. R. China.
Email:
ZhaoG@ustc.edu.cn.

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