Volume 30, No. 5 September/October 2009 ISSN 0143-2044
	INSECT COLD TOLERANCE AND REPAIR OF CHILL-INJURY AT FLUCTUATING THERMAL REGIMES: ROLE OF 70 KDA HEAT SHOCK PROTEIN EXPRESSION Michaela Tollarová-Borovanská, Lisa Lalouette  and Vladimír Koštál Suzuki	312-319
	DEVELOPMENT OF ALTERNATIVE PLANT VITRIFICATION SOLUTIONS IN DROPLET-VITRIFICATION PROCEDURES Haeng-Hoon Kim, Yoon-Geol Lee, Dong-Jin Shin, Ho-Cheol Ko, Jae-Gyun Gwag, Eun-Gi Cho and Florent Engelmann	320-334
	A DEVICE TO MEASURE OXYGEN CONSUMPTION DURING THE HYPOTHERMIC PERFUSION OF THE LIVER Joaquín V. Rodriguez, María Belen Federico, María Dolores Pizarro, Edgardo E. Guibert, Alejandra B. Quintana and Angel L. Scandizzi	335-346
	CRYOPRESERVATION OF IN VITRO GROWN SHOOT TIPS AND APICAL MERISTEMS OF THE FORAGE LEGUME ARACHIS PINTOI Hebe Y. Rey, Mirta Faloci, Ricardo Medina, Natalia Dolce, Luis Mroginski and Florent Engelmann	347-358
	CRYOPRESERVATION OF OLIVE EMBRYOGENIC CULTURES C. Sánchez-Romero, R. Swennen and B. Panis	359-372
	EFFECTS OF CRYOPROTECTANT ON THE EMBRYOS OF BANDED CORAL SHRIMP (STENOPUS HISPIDUS); PRELIMINARY STUDIES TO ESTABLISH FREEZING PROTOCOLS S. Tsai and C. Lin	373-381
	EU COST Action 871 Abstracts 2009 Royal Botanic Gardens, Kew, Wakehurst Place, UK	382-397

Top of page

CryoLetters 30 (5), 312-319 (2009)
© CryoLetters, businessoffice@cryoletters.org

INSECT COLD TOLERANCE AND REPAIR OF CHILL-INJURY AT FLUCTUATING THERMAL REGIMES: ROLE OF 70 KDA HEAT SHOCK PROTEIN EXPRESSION

Michaela Tollarová-Borovanská ^{1, 2}, Lisa Lalouette ³ and Vladimír Koštál ^{1, 2}*

¹ Biology Centre ASCR, Institute of Entomology, České Budějovice, Czech Republic
² University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic
³ UMR CNRS 6553, University of Rennes 1, Rennes, France
*Correspondence author email: kostal@entu.cas.cz

Abstract

Expression of heat shock proteins has been proposed as an underlying mechanism of increased cold tolerance in insects exposed to fluctuating thermal regimes (FTRs) in comparison to constant low temperatures (CLTs). We found that the levels of Pahsp70 mRNA increase by up to 3 orders in the linden bugs, Pyrrhocoris apterus exposed to FTR -5ºC (22h)/ 25ºC (2h). The 2h-long warm pulses, however, were not sufficient for accumulation of PaHSP70 protein and thus no significant difference in expression of PaHSP70 protein was detected between FTR and CLT regimes. Hence, we conclude that the accumulation of PaHSP70 protein is not the mechanism underlying the increased cold tolerance in P. apterus at the particular FTR used in this study. The relevance of some other possible mechanisms is discussed.

Keywords: insect; cold tolerance; heat shock proteins; fluctuating thermal regime

Top of page

CryoLetters 30 (5), 320-334 (2009)
© CryoLetters, businessoffice@cryoletters.org

DEVELOPMENT OF ALTERNATIVE PLANT VITRIFICATION SOLUTIONS IN DROPLET-VITRIFICATION PROCEDURES

Haeng-Hoon Kim^1*, Yoon-Geol Lee¹, Dong-Jin Shin¹, Ho-Cheol Ko¹, Jae-Gyun Gwag¹, Eun-Gi Cho¹ and Florent Engelmann^{2, 3}

¹National Academy of Agricultural Science, RDA, Suwon 441-707, Korea.
²Institut de recherche pour le développement (IRD), UMR DIAPC, BP 64501, 34394 Montpellier cedex 5, France.
³Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Correspondence author email: hkim@rda.go.kr

Abstract

This study aimed at developing alternative vitrification solutions, modified either from the original PVS2 vitrification solution by increasing glycerol and sucrose and/or decreasing dimethylsulfoxide and ethylene glycol concentration, or from the original PVS3 vitrification solution by decreasing glycerol and sucrose concentration. The application of these vitrification solutions to two model species, i.e. garlic and chrysanthemum in a droplet-vitrification procedure, revealed that PVS3 and variants were superior to PVS2 and variants and that most PVS2 variants were comparable to the original PVS2. Both species were sensitive to chemical toxicity of permeating cryoprotectants and chrysanthemum was also sensitive to osmotic stress. The lower recovery of cryopreserved garlic shoot apices dehydrated with PVS2 and variants compared with those dehydrated with PVS3 and variants seemed attributed to cytotoxicity of the vitrification solutions tested as well as to insufficient protection against freezing injury. Chrysanthemum shoot tips were very sensitive to both chemical toxicity and osmotic stress and therefore, induction of cytotoxity tolerance during preconditioning was required for successful cryopreservation. The present study revealed that some of the PVS2 variants tested which have increased glycerol and sucrose and/or decreased dimethylsulfoxide and ethylene glycol concentration can be applied when explants are of medium size, tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

Keywords: chrysanthemum, dehydration, droplet-vitrification, garlic, vitrification solution

Top of page

CryoLetters 30 (5), 335-346 (2009)
© CryoLetters, businessoffice@cryoletters.org

A DEVICE TO MEASURE OXYGEN CONSUMPTION DURING THE HYPOTHERMIC PERFUSION OF THE LIVER

Joaquín V. Rodriguez^**, María Belen Federico^*, María Dolores Pizarro^*, Edgardo E. Guibert^†, Alejandra B. Quintana^†† and Angel L. Scandizzi^*

^*Farmacología, ^††Morfología, Departamento de Ciencias Fisiológicas, ^†Biología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531-S2002LRK Rosario, Argentina.
**Address correspondence and reprints requests to Joaquin V. Rodriguez, Farmacologia, Depto Cs. Fisiologicas, Facultad de Cs. Bioquimicas y Farmaceuticas, UNR, Suipacha 531, S2002LRK Rosario, Argentina.
Corresponding author email: jrodrig@fbioyf.unr.edu.ar

Abstract

Abstract. This work deals with the construction and performance of a device designed to measure the oxygen consumption by the liver during hypothermic perfusion in the rat model. Due to its simple design and the utilization of standard materials, it could serve to determine the role of oxygenation during hypothermic perfusion of the liver. The system consists of a reservoir containing the preservation solution, a peristaltic pump and an internal oxygenator made of silicone tube. A five ports manifold connects the circulation to the liver (inflow), to a hydrostatic manometer and to two sample ports; the liver outflow and temperature sensor or gas calibration. Finally the exit port connects the circulation fluid with an oxygen electrode. The preservation solution is pumped through the liver at a constant pressure (77  15 mmH₂O) and a perfusion flow of 0.39 – 0.49 mL.min^-1.g liver^-1 . To test the system, two to four hours perfusion experiments were performed, at temperatures of 5 and 10C. Two preservation solutions were evaluated: Custodiol and Bes-Gluconate-Sucrose. The solubility of oxygen in the preservation solutions was determined, and the oxygen consumption by preserved rat livers was measured.

Keywords: liver, cold storage, hypothermic perfusion, oxygen consumption

Top of page

CryoLetters 30 (5), 347-358 (2009)
© CryoLetters, businessoffice@cryoletters.org

CRYOPRESERVATION OF IN VITRO GROWN SHOOT TIPS AND APICAL MERISTEMS OF THE FORAGE LEGUME ARACHIS PINTOI

Hebe Y. Rey¹*, Mirta Faloci¹, Ricardo Medina¹, Natalia Dolce¹, Luis Mroginski¹ and Florent Engelmann^{2, 3}

¹Instituto de Botánica del Nordeste (IBONE), CONICET – UNNE, Facultad de Ciencias Agrarias (UNNE), C.C. 209, 3400 Corrientes, Argentina.
²IRD, UMR DIAPC, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France.
³Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Corresponding author  email: heberey@agr.unne.edu.ar

Abstract

A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23% moisture content (fresh weight basis). Explants were frozen using slow cooling (1ºC min^-1 from 25ºC to -30ºC followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 μM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53% and 56% of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16% of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.

Keywords: apical meristems, cryopreservation, encapsulation-dehydration, genetic stability, shoot tips.

Top of page

CryoLetters 30 (5), 359-372 (2009)
© CryoLetters, businessoffice@cryoletters.org

CRYOPRESERVATION OF OLIVE EMBRYOGENIC CULTURES

C. Sánchez-Romero^1a*, R. Swennen² and B. Panis²

¹IFAPA Centro de Churriana, Cortijo de la Cruz s/n, 29140 Churriana-Málaga, SPAIN.
²Laboratory of Tropical Crop Improvement, KU Leuven, Kasteelpark Arenberg 13, 3001 Leuven, BELGIUM.
^aCurrent address: Dpto. Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, SPAIN
*Corresponding author  email: c.sanchez@uma.es

Abstract

The aim of this work was to optimize a protocol for the cryopreservation of embryogenic cultures of olive (Olea europaea L.). Exposure time to loading solution and PVS2 significantly influenced the regrowth rate of both organized and non-organized tissues. Organized tissues were more sensitive to prolonged treatments with vitrification solutions compared to non-organized tissues. Three cryopreservation protocols were compared using non-organized tissues: the "classical" vitrification protocol, an ultra-fast freezing method using droplet vitrification on aluminium foil strips and a "classical" slow freezing method (1^oC min^-1). The best results were obtained using the droplet vitrification method after a 60 min dehydration period with PVS2. Under these conditions, all cryopreserved cultures showed renewed embryogenesis six weeks after thawing. A long-term (7-8 weeks) sucrose preculture had a significant effect on the initial response of the cultures, allowing particularly to protect cells against the toxic effects of the vitrification solution.

Keywords: droplet vitrification, embryogenic tissue, long-term preculture, olive, slow freezing, vitrification-based protocols

Top of page

CryoLetters 30 (5), 373-381 (2009)
© CryoLetters, businessoffice@cryoletters.org

EFFECTS OF CRYOPROTECTANT ON THE EMBRYOS OF BANDED CORAL SHRIMP (STENOPUS HISPIDUS); PRELIMINARY STUDIES TO ESTABLISH FREEZING PROTOCOLS

S. Tsai and C. Lin*

National Museum of Marine Biology & Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
*Correspondence author e-mail: chiahsin@nmmba.gov.tw

Abstract

The addition of cryoprotectants is a necessary step in cryopreservation procedures because they can minimize cellular injury during cryopreservation. Toxicity of cryoprotectant depends on the type, concentration, temperature and exposure period. The aim of this study was to investigate the toxicity of cryoprotectant to embryos of banded coral shrimp (Stenopus hispidus) in order to inform the development of a cryopreservation protocol. Three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) embryos were selected and exposed to different concentration of cryoprotectants (0.25M-5M) for an equilibration period of 10, 20 or 30 min at room temperature. Hatching percentage indicated that the toxicity of tested cryoprotectants increased in the order of methanol, ethylene glycol (EG), dimethyl sulphoxide (DMSO), glycerol and dimethylacetamide (DMA). The No Observed Effect Concentrations (NOECs) for eye-formation stage embryos were 1M, 0.25M, 0.25M, 0.25M and 0.25M respectively after 10 min incubation whilst the NOECs for heart beat and pre-hatch stage embryos were 1M, 0.5M, 0.5M, 0.5M and 0.25M respectively. Pre-hatch stage embryos appeared to be more tolerant to cryoprotectant toxicity than eye-formation and heart beat stage embryos.

Keywords: cryoprotectant, banded coral shrimp (Stenopus hispidus), embryo, methanol, dimethylacetamide (DMA)

Home  Aims and Scope  Abstracts  Perspectives  Editorial Board  Info for Authors  Subscriptions  Links