CryoLetters

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

CRYOPRESERVATION OF PLUMULAR EXPLANTS OF COCONUT (Cocos nucifera L.) TO SUPPORT PROGRAMMES FOR MASS CLONAL PROPAGATION THROUGH SOMATIC EMBRYOGENESIS

Roland Hornung¹, Raphaël Domas^{1, 2} and Paul T. Lynch^3*

¹Plant Biotechnology Laboratories, Imperial College at Wye, T.H. Huxley School, Wye, Ashford, Kent, TN25 5AH, UK
²Current address: rue Le Nôtre, Angers, 49045 France.
³Division of Biological Sciences, School of Environmental and Applied Sciences, University of Derby, Kedleston Road, Derby DE22 1GB, UK
* To whom correspondence should be addressed (e.mail: p.t.lynch@derby.ac.uk).

Summary

Callus growth has been observed from plumules of ecotype Laguna Tall after cryopreservation using an encapsulation/dehydration protocol.  Sucrose preculture treatment and silica gel dehydration both significantly influenced the frequency of callus formation from non-frozen and frozen plumules. The greatest frequency of post-thaw callus growth occurred after incubation of the encapsulated plumules for 72- 96 h in medium containing 0.75 M sucrose followed by desiccation over silica gel for 7 – 8 h down to approximately 30% moisture content (fresh weight basis).  Freezing and thawing were carried out rapidly. Post-thaw recovery rates in excess of 80% were recorded.

Keywords: coconut, plumule, somatic embryogenesis, cryopreservation, encapsulation/dehydration

PREGROWTH-DESICCATION: A SIMPLE AND EFFICIENT PROCEDURE FOR THE CRYOPRESERVATION OF RICE (Oryza sativa L.) EMBRYOGENIC SUSPENSION CELLS

Yi-Xiang Zhang, Jun-Hui Wang*, Hong-Wu Bian, Mu-Yuan Zhu

College of Life Sciences, Zhejiang University, Hangzhou 310012, China
* Corresponding author, E-mail: junhuiwang@cls.zju.edu.cn

Summary

Rice embryogenic suspension cells were successfully cryopreserved by a pregrowth-desiccation procedure. Cells were precultured in liquid AA medium containing 0.175 mol/L sucrose for 3 d and then in liquid AA medium containing 0.4 mol/L sorbitol for 1 d. After air-drying for about 20 h to a water content of 10 %, the cells were placed into cryotubes and quenched into liquid nitrogen. Using this pregrowth-desiccation procedure, a survival rate of 96±6 % (TTC reduction assay) or 100 % (cell clump regrowth) was achieved. Cryostored cells revived very quickly during the recovery culture and they retained the ability to regenerate fertile plants. In conclusion, air-drying, a method usually employed in cryopreservation of seeds or shoot tips, can be used as a simple and efficient procedure for the cryopreservation of precultured rice suspension cells.

Key words: cryopreservation, pregrowth, desiccation, rice, embryogenic suspension cells

THE EFFECTS OF FREEZING AND CRYOPROTECTANT EXPOSURE ON ADENYLATE CYCLASE ACTIVITY IN CELL MEMBRANES OF BOVINE THYROID GLAND AND ADRENAL CORTEX

L.G Karpenko*, N.F Gubina, V.A Schirova and A.S.Kaprelyants

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 61015, 23, Pereyaslavskaya str., Kharkov, Ukraine

Summary

We present data on the effect of freezing with or without cryoprotectant (CPA) dimtethyl suphoxide  (Me₂SO), as well as effects of exposure to CPA alone on the activity of adenylate cyclase in cellular membranes of thyroid glands and adrenal cortex. Our results show that freezing without cryoprotectant increases unstimulated adenylate cyclase activity and decreases sensitivity to specific regulators. CPA at a concentration of 5% for adenylate cyclase in adrenal cortex tissue and of 10% in thyroid gland allowed preservation of the best responses to specific receptor agonists.

Keywords: adenylate cyclase, thyroid gland, adrenal cortex, freezing, cryoprotectant exposure; Me₂SO

USE OF SPERM STORED UNDER HYPOTHERMIC CONDITIONS FOR REINSEMINATION OF UNFERTILIZED OOCYTES IN AN IVF PROGRAMME

V.I. Grischenko*, M.P. Petrushko, V.I. Pinyayev, and I.V. Terpyachaya

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 23, Pereyaslavskaya str., 61015 Kharkov, Ukraine

Summary

The work describes the data on the selection of optimal hypothermic storage for  progressively moving fraction of human spermatozoa isolated from ejaculate. Results were analysed by assessing sperm motility, oocyte fertilization rate, and embryonic development  after re-insemination of unfertilized oocytes by spermatozoa stored under hypothermic conditions.

Keywords: human spermatozoa, hypothermic storage, oocyte re-insemination.

MEMBRANE DAMAGE OCCURS DURING THE FORMATION OF INTRACELLULAR ICE

Jason P. Acker* and Locksley E. McGann

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada T6G 2R8

Summary

Current theories on both the formation of intracellular ice (IIF) and the mechanisms by which it damages cells all implicate the plasma membrane. While it has been well documented that following IIF post-thaw damage to the plasma membrane occurs, it is not known whether this damage occurs during freezing or thawing. By directly monitoring the diffusion of a membrane-impermeable fluorescent stain into V-79W fibroblasts, the integrity of the plasma membrane during freezing and thawing was correlated with the incidence of IIF.  While this study presents evidence that membrane damage occurs during the formation of intracellular ice in cell suspensions, single attached cells and confluent monolayers, we cannot conclude whether IIF is the cause or result of membrane damage.

Keywords: cryoinjury, V-79W fibroblasts, membrane damage, intracellular ice

BIOMETRIC ANALYSIS OF PHENOTYPIC CHARACTERS OF POTATO SHOOT-TIPS RECOVERED FROM TISSUE CULTURE, DIMETHYL SULPHOXIDE TREATMENT AND CRYOPRESERVATION

K.Harding¹* and H.Staines²

¹DAMAR, Drum Road, Cuparmuir, Cupar, Fife,  KY15 5RJ Scotland UK.
email: DAMAR @tinyworld.co.uk
²Division of Mathematics and Statistics, School of Computing, University of Abertay Dundee, Bell Street, Dundee DD1 1HG Scotland UK
*Corresponding author.

Summary

A biometric approach partly involving principal component analysis has been used to examine the changes in phenotypic characters of shoot-tip derived potato plants recovered from tissue culture, dimethyl sulphoxide (DMSO) and cryopreservation in comparison with field-grown tuber-derived plants. There were significant differences in the quantitative characters height and tuber weight in the cryopreserved plants compared to those derived from DMSO treatment and tissue culture. These three experimental groups were shown to significantly differ from field-grown plants in the characters, tuber weight followed by height and length of petiole. The use of biometrics as an analytical approach in genetic stability assessments of plants recovered from cryopreservation to relatively small sample sizes is discussed.

Keywords: S. tuberosum, cryopreservation, phenotype, biometric analysis.

THE USE OF FLOW CYTOMETRY TO ASSESS MEMBRANE STABILITY IN FRESH AND CRYOPRESERVED TROUT SPERMATOZOA

E. Cabrita, F. Martínez, M. Real, R. Alvarez and M.P. Herráez*

Department of Cell Biology, Faculty of Biology, University of Leon, 24071 Leon, Spain

Summary

  In this approach flow cytometry combined with PI was used as an alternative method to determined cell membrane integrity and osmotic fragility in trout spermatozoa. Milt was diluted 1:3 in a extender containing either 7% Me₂SO or 7% Me₂SO plus egg yolk-BSA and frozen in 0.5ml French straws on a rack that floated 2cm over the surface of liquid nitrogen. Cell integrity and membrane resistance to hyposmotic shock was analyzed before and after freezing/thawing. To test cell resistance to hyposmotic shock, sperm was subjected to hypo- and isosmotic solutions (10, 100, 300mOsm/Kg) and cell integrity was analyzed through a time course (30sec, 2, 5, 10 and 15min) using propidium iodide and flow cytometry. This procedure allowed a rapid and accurate identification of different cell subpopulations according to their resistance to hyposmotic shock and, in this way, provides a method to evaluate the sub-lethal damage caused by cryopreservation. The proportion of extremely sensitive cells in fresh spermatozoa (2%) increased after cryopreservation: 30% without stabilizers, and 19% after the addition of egg yolk-BSA, demonstrating that external cryoprotectants significantly improved the resistance to the osmotic stress.

 Keywords: flow cytometry, sperm membrane stability, trout sperm, cryopreservation, membrane stabilizers.

Abstracts

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