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Abstracts: CryoLetters 31 (3), 2010

 

 

Volume 31, No. 3 May/June 2010

ISSN 0143-2044

 

 


Recovery and characterisation of hybrid FIRS (Abies alba x A. cephalonica, Abies alba x A. numidica) embryogenic tissues after cryopreservation
Terezia Salaj, Ildiko Matusikova, Bart Panis, Rony Swennen and Jan Salaj

206-217

 

 


Induction of phospho-thr-172 ampk in 3t3-l1 adipocytes exposed to cold or treated with anisomycin, mithramycin a, and ionic compounds
Yasuhito Ohsaka, Hoyoku Nishino and Yasuyuki Nomura

218-229

 

 


Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification
M Guan, D M Rawson and T Zhang

230-238

 

 


Ice-active proteins from new zealand snow tussocks, Chionochloa macra and C. rigida
Wharton D A, Selvanesan L and Marshall C J

239-248

 

 


Physiological, biochemical and molecular characteristics of cryopreserved Hypericum perforatum L. shoot tips
Matúš Skyba, Martina Urbanová, Veneta Kapchina-Toteva, Ján Košuth, Keith Harding and Eva Čellárová

249-260

 

 


Effect of cryopreservation protocols on the phenotypic stability of yeast
Pushpa Gujjari, Tamara Muldrow and Jianlong Jim Z

261-267

 

 


An improved pollen collection and cryopreservation method for highly recalcitrant tropical fruit species of mango (Mangifera indica L.) and litchi (litchi chinensis sonn.)
Rekha Chaudhury, S K Malik and S R

268-278

 

 


A universal self-adaptive time-varying function for extracellular concentration during osmotic shift for curve-fitting permeability coefficients of cell membrane
Gang Zhao

 279-290

 

 

 

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CryoLetters 31 (3), 206-217 (2010)
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RECOVERY AND CHARACTERISTICS OF HYBRID FIRS (Abies alba x A. cephalonica, Abies alba x A. numidica) EMBRYOGENIC TISSUES AFTER CRYOPRESERVATION

Terezia Salaj1*, Ildiko Matusikova1, 3, Bart Panis2, Rony Swennen2 and Jan Salaj1

1Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, PO Box 39A, 95007 Nitra, Slovak Republic.
2Laboratory of Tropical Crop Improvement, K.U. Leuven, Kastelpark Arenberg 13, B-3001 Leuven, Belgium
3Department of Health and Environment/Bioresources, Austrian Research Centres GmbH-ARC, 2444 Seibersdorf, Austria
*Corresponding author e-mail:
nrgrtesa@savba.sk

Abstract

Embryogenic tissues of hybrid firs were cryopreserved using a slow freezing protocol. The procedure involved preculture of tissues for 24, 48 or 72 h in media with different sorbitol concentrations (0.4 or 0.8 M) and addition of 5% (v/v) DMSO as cryoprotectant. The cell lines tested withstood cryopreservation, even though tissue regrowth after thawing was dependent on treatment and cell line. For cell line AN72, regrowth was 100% for all experimental conditions tested. With cell line AC78, regrowth was 100% except after shorter pretreatment durations, which produced 83% and 86% regrowth for 0.4 M and 0.8 M sorbitol pretreatment, respectively. Cell lines AC1 and AC4 were more sensitive to cryopreservation with 37.5 to 100% regrowth, respectively. Growth parameters evaluated 3 months after cryopreservation showed cell line and treatments effects. In most cases, cryopreservation had no negative effect on growth of tissues. Statistically significant differences in fresh mass accumulation were found for four samples out of 24 investigated, although growth increase of these tissues still reached 79.4-84.6%, compared with non-cryopreserved ones (100% increase). Maturation capacity and genetic fidelity were studied in tissues whose growth was not negatively influenced by cryopreservation. Maturation capacity of embryogenic tissues cryopreserved using the optimal protocol was comparable to that of non-frozen controls. RAPD analysis of 88 genomic regions per cell line did not reveal any changes in genetic fidelity of cryopreserved tissues compared to non-cryopreserved controls.

Keywords: Abies hybrids, cryopreservation, embryogenic tissues, plantlet regeneration, RAPD analyses

 

 

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CryoLetters 31 (3), 218-229 (2010)
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INDUCTION OF PHOSPHO-THR-172 AMPK IN 3T3-L1 ADIPOCYTES EXPOSED TO COLD OR TREATED WITH ANISOMYCIN, MITHRAMYCIN A, AND IONIC COMPOUNDS

Yasuhito Ohsaka1,3,*, Hoyoku Nishino2,3 and Yasuyuki Nomura4

1Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan
2Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan
3Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
4Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama, Kanagawa 245-0066, Japan
*Corresponding author  e-mail:
y-ohsaka@cis.ac.jp

Abstract

Cold exposure induces cellular responses, including subcellular molecule expression and transport responses, similar to those stimulated by insulin in 3T3-L1 (L1) adipocytes. The transport response is induced in L1 adipocytes treated with translation inhibitors. We examined the level of phospho-Thr-172 AMPK (an active form of AMPK, a known energy-state sensor) in L1 adipocytes exposed to different temperatures of 4–37°C or stressors, including chemical inhibitors and activators. The phospho-AMPK level increased in cold-exposed cells and their subcellular fractions and decreased after rewarming and serum depletion. The phospho-molecule was also induced by anisomycin, which induces protein kinase activation and translation inhibition; mithramycin A, an inhibitor of transcription factor binding; and ionic compounds, which stimulate molecular signaling and alter several gene expression. These results indicate that temperature responses are mimicked by metabolic stressors through phospho-molecule alteration. Our results provide possible clues for clarifying the mechanisms underlying cold responses in L1 adipocytes.

Keywords: AMP-activated protein kinase, cold, Thr-172, 3T3-L1 adipocytes

 

 

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CryoLetters 31 (3), 230-238 (2010)
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CRYOPRESERVATION OF ZEBRAFISH (Danio Rerio) OOCYTES BY VITRIFICATION

M. Guan, D. M. Rawson and T. Zhang*

LIRANS Institute of Research in the Applied Natural Science, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK
*Corresponding author email:
tiantian.zhang@beds.ac.uk

Abstract

Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30min at 22ºC post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.

Keywords: zebrafish, vitrification, trypan blue staining

 

 

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CryoLetters 31 (3), 239-248 (2010)
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ICEACTIVE PROTEINS FROM NEW ZEALAND SNOW TUSSOCKS, Chionochloa macra AND C. rigida

Wharton, D.A.1*, Selvanesan, L.2 and Marshall, C.J.2

Department of Zoology1 and Biochemistry2, University of Otago, P.O. Box 56, Dunedin, New Zealand.
*Corresponding author email:
david.wharton@stonebow.otago.ac.nz

Abstract

The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process.

Keywords: ice nucleators; recrystallization inhibition; thermal hysteresis; antifreeze protein; fog collection.

 

 

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CryoLetters 31 (3), 249-260 (2010)
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PHYSIOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERISTICS OF CRYOPRESERVED Hypericum Perforatum L. SHOOT TIPS

Matúš Skyba1, Martina Urbanová1, Veneta Kapchina-Toteva2, Ján Košuth1, Keith Harding3 and Eva Čellárová1*

1Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University, Mánesova 23, 04154 Košice, Slovakia
2Faculty of Biology, University of Sofia, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria
3Research Scientists, Conservation & Biotechnology, Damar, Drum Road, Cupar, Fife, Scotland KY15 5RJ, UK
*Corresponding author  e-mail:
eva.cellarova@upjs.sk

Abstract

Hypericum perforatum L. in vitro cultured shoot tips were characterised at the physiological, biochemical and molecular levels following recovery from cryogenic treatment using the plant vitrification solutions PVS2 and PVS3. This comparative study revealed an increase in recovery and regrowth of explants cryoprotected with PVS3. Among the physiological markers only lipid peroxidation in the regenerants treated with PVS2 significantly increased indicating membrane damage. Genotype-specific interactions were found in most characteristics studied, with some variation detected within control and cryopreserved samples. Analyses of metabolite biosynthesis and genetic stability showed no significant differences in hypericin content, RAPD and minisatellite amplification profiles between PVS2- and PVS3-treated explants. This study demonstrates and discusses the criteria selective for PVS3 to improve the cryopreservation of H. perforatum L.

Keywords: cryoprotection, hypericin, PVS2, PVS3, RAPD, VNTR, vitrification

Abbreviations: ABA abscisic acid; BA 6-benzylaminopurine; bp base pairs; DMSO dimethylsulphoxide; FW fresh weight; H2O2 hydrogen peroxide; KI potassium iodide; LN liquid nitrogen; LS loading solution; MDA malonyldialdehyde; M.wt. Molecular weight; PVS plant vitrification solution; RAPD random amplified polymorphic DNA; ROS reactive oxygen species; TBA thiobarbituric acid; TCA trichloroacetic acid; VNTR variable number of tandem repeats

 

 

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CryoLetters 31 (3), 261-267 (2010)
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EFFECT OF CRYOPRESERVATION PROTOCOLS ON THE PHENOTYPIC STABILITY OF YEAST

Pushpa Gujjari, Tamara Muldrow and Jianlong Jim Zhou*

*Mycology Program, American Type Culture Collection (ATCC), 10801 University Blvd, Manassas, Virginia 20110, USA;
Corresponding author email:
jzhou@atcc.org

Abstract

Eight cryopreservation protocols were assessed for their effects on the viability and phenotypic stability of the yeast Saccharomyces cerevisiae during a five-year study. It is found that viability and phenotypic features have remained largely unchanged when the yeast was preserved in glycerol, dimethyl sulphoxide, or sucrose at 80°C or in liquid nitrogen. When sorbitol was used as a cryoprotectant, yeast cells frozen and stored at 80°C manifested great decreases in viability after six months in storage and concomitantly large fluctuations in the rate of the trp1 auxotrophic reversion. This phenotypic reversion was stable passage after passage. Such a degree of phenotypic fluctuations, however, was not observed for yeast cells preserved in the same sorbitol solution that went through a controlled freezing program and were subsequently stored in liquid nitrogen. These results indicate that some combinations of cryoprotective agent, freezing program, and storage temperature disturb biomaterials more profoundly during cryopreservation and imply a genetic basis of this phenotypic change.

Keywords: Cryopreservation, cryoprotectant, phenotypic stability, genetic, yeast 

 

 

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CryoLetters 31 (3), 268-278 (2010)
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AN IMPROVED POLLEN COLLECTION AND CRYOPRESERVATION METHOD FOR HIGHLY RECALCITRANT TROPICAL FRUIT SPECIES OF MANGO  (Mangifera indica L.) AND LITCHI (Litchi chinensis Sonn.)

Rekha Chaudhury1*, S.K. Malik1 and S. Rajan 2

1Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, India
2 Central Institute for Subtropical Horticulture, Rehmankhera, Lucknow, Uttar Pradesh, India
*Corresponding author email:
rekha@ nbpgr.ernet.in

Abstract

An improved method for pollen collection from freshly dehiscing anthers of mango (Mangifera indica L.) and litchi (Litchi chinensis Sonn.) using the organic solvent cyclohexane has been devised. Using this method pollen quantity sufficient for large scale pollinations could be collected and stored for future use. Transport of pollen in viable conditions over long distances, from site of collection (field genebank) to cryolab was successfully devised for both these fruit species. Cryopreservation was successfully applied to achieve long-term pollen storage over periods of up to four years. Pollen viability was tested using in vitro germination, the fluorochromatic reaction (FCR) method and by fruit set following field pollination. On retesting, four year cryostored pollen of different mango and litchi varieties showed high percentage viability as good as fresh control pollens. Pollens of more than 180 cultivars of mango and 19 cultivars of litchi have been stored in the cryogenebank using the technology developed, thus facilitating breeding programmes over the long-term.  

Keywords: mango, litchi, cryopreservation, pollen, viability, cyclohexane

 

 

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CryoLetters 31 (3), 279-290 (2010)
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A UNIVERSAL SELF-ADAPTIVE TIME-VARYING FUNCTION FOR EXTRACELLULAR CONCENTRATION DURING OSMOTIC SHIFT FOR CURVE-FITTING PERMEABILITY COEFFICIENTS OF CELL MEMBRANE

Gang Zhao1,2*

1 Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, P.R. China.
2 Department of Mechanical Engineering Science, Kyushu University, Fukuoka 819-0395, Japan.
*Corresponding author  e-mail:
zhaog@ustc.edu.cn

Abstract

A universal self-adaptive time-varying function of extracellular concentration history during osmotic shift for measuring cell membrane permeability was presented in this study. The feasibility and accuracy of the assumed function were verified based on the experimental data obtained from the microperfusion chamber method. It was found that the assumed function could always give out the very satisfactory coefficient of determination, and there were no significant differences between the hydraulic conductivity values fitted using the laser interferometer measured extracellular concentration profile and the predicted one by the assumed piecewise function (student's t test, p>0.05). Due to the adaptive feature of the assumed function for the concentration of extracellular solution, the function was suggest to be used for all the similar studies for measurement of cell membrane permeability by osmotic shift.

Keywords: hydraulic conductivity, curve fitting, extracellular concentration, osmotic shift

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