CRYOPRESERVATION OF PLUMULAR EXPLANTS OF COCONUT (Cocos nucifera L.) TO SUPPORT PROGRAMMES FOR MASS CLONAL PROPAGATION THROUGH SOMATIC EMBRYOGENESIS
Roland Hornung1, Raphaël Domas1, 2 and Paul T. Lynch3*
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: email@example.com).
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: firstname.lastname@example.org
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
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
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
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
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
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.