CryoLetters

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

CryoLetters 24, 1 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EDITORIAL - GROWTH AND SUSTAINABILITY

CryoLetters 24, 5-16 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A SIMPLE CAPACITIVE CELL FOR THE MEASUREMENT OF LIQUIDS DIELECTRIC CONSTANT UNDER TRANSIENT THERMAL CONDITIONS

A. Baudot* and J.L. Bret

Centre de Recherches sur les Très Basses Températures, C.N.R.S. - BP 166 - 38042 Grenoble Cedex 9, France. *E-mail: baudot@grenoble.cnrs.fr

Abstract

A simple device for the measurement of the complex dielectric permittivity of liquids in various thermodynamic states has been developed. It uses a cylindrical aluminium capacitor of a type currently applied in tuning antenna circuits. The capacitor is filled with the liquid solution under study. A comparison of its capacity is made with that of the nitrogen filled capacitor tested under the same thermal conditions. This comparison allows the determination of the real and imaginary part of the solutions permittivity as a function of temperature (between 150 and 300 K) and frequency (between 100 Hz to 2 MHz). After validating the technique with pure glycerol and pure 1,2-propanediol, spectroscopic measurements have been undertaken on pure and diluted 1,2-propanediol in water. Due to the low heat capacity and the high thermal conductivity of the capacitor, cooling rates of 40 K/min have been achieved inside the solution, allowing measurements in the supercooled liquid and vitreous states. Results are presented and discussed in terms of relaxation and the physical states of the sample. By selecting the required thermal conditions, this device permits the observation of thermal transitions, such as ice crystallisation, and measurements to be conducted in the unstable supercooled liquid state. These measurements are necessary in the development of an effective electromagnetic warming device for vitrified cryoprotective solutions.

Keywords: dielectric permittivity, low frequency, glycerol, 1,2-propanediol, vitrification, ice crystallisation, supercooled liquid state.

CryoLetters 24, 17-26 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

SEED CRYOPRESERVATION AND LONGEVITY OF TWO Salix HYBRIDS

Christopher B. Wood^1*, Hugh W. Pritchard¹ and Kevin Lindegaard²

¹Seed Conservation Department, Royal Botanic Gardens, Kew, Wellcome Trust Millennium Building, Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK
²Institute of Arable Crop Research (IACR), Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS4H 9AF, UK
^*For correspondence: c.wood@rbgkew.org.uk

Abstract

The effects of desiccation and storage temperature on the viability and longevity of willow seeds was investigated using two hybrids, Salix rehderiana x (Salix x capreola) [cross 458] and Salix x sericans x Salix viminalis [cross 512]. Freshly harvested seed of both crosses survived silica gel drying down to c. 3 to 5 % moisture content. Hybrid 458 seed stored in liquid nitrogen (-196C) for 3 d retained viability when equilibrated to 45 % RH (pre-storage), showed slightly reduced survival at 65 % RH and exhibited no survival at 82 % RH. The level of survival after 68 d for seeds pre-equilibrated to either c. 10 or 65 % RH (5 or 10 % moisture) and stored at four temperatures was -196C > -20C > 2C > 16C. At all temperatures, drier seed stored better than wetter seed. For hybrid 512, seed longevity at 20C > 40C > 60C, and a 10 % fall in pre-storage seed RH resulted in a c. 2-fold increase in longevity at each storage temperature. The response of hybrid willow seeds to desiccation and cooling raises possibilities for the long-term seed conservation of Salix species by cryopreservation.

Keywords: Salix, orthodox seed, cryopreservation, desiccation, longevity.

CryoLetters 24, 27-32 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

THE INCIDENCE OF MITOTIC ABNORMALITIES IN CRYOPRESERVED EIGHT-CELL EARLY AND COMPACTED MOUSE EMBRYOS

O.B. Khromenkova^1*, G.V. Zhernoklev², G.F. Zhegunov¹ and V.I. Grischenko¹

¹Kharkov State Medical University, 4, Lenin ave, 61022 Kharkov, Ukraine
²Biotechnological Center, Kulinichi, 62040 Kharkov region, Ukraine
e-mail: khromenkova@mail.ru

Abstract

The effects of slow freezing–rapid thawing on viability and chromosomal complement of eight-cell early and eight-cell compacted mouse embryos were investigated. The abnormalities connected with damage to the mitotic apparatus, and with chromosome damage were investigated in cryopreserved embryos by cytological analysis. The embryos were preserved using 1M glycerol as cryoprotectant and a slow cooling regime to -30ºC before transfer to liquid nitrogen. The proportion of mitotic abnormalities in compacted embryos was significantly higher (13.1%) than in early embryos (5.9%) and unfrozen control embryos (3.9%) in these studies performed after thawing. This was reflected after cryopreservation by a reduced viability of the embryos as judged by culture to the hatching blastocyst stage – compacted 8-cell embryos (71.84.7%) versus controls (78.45.9%) and cryopreserved early-stage 8-cell embryos (86.14.0%) – p<0.05 in each case. However, aneuploidy rates were low in all groups in both fresh and cryopreserved embryos (around 3%),.

Keywords: preimplantation embryos, cryopreservation, mitotic abnormality, aneuploidy.

CryoLetters 24, 33-42 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

RECOVERY OF POTATO APICES AFTER SEVERAL YEARS OF STORAGE IN LIQUID NITROGEN

G. Mix-Wagner¹*, H.M. Schumacher² and R.J. Cross³

¹Institute of Crop and Grassland Science, Federal Agricultural Research Centre (FAL), Bundesallee 50, 38116 Braunschweig, Germany, gunda.mix@fal.de
²Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1 b, 38124 Braunschweig, Germany, mas@dsmz.de
³New Zealand Institute for Crop & Food Research Limited, Private Bag 4704, Christchurch, New Zealand, crossr@crop.cri.nz
* Corresponding author

Abstract

   A method for the systematic cryopreservation of potato apices was developed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the Institute for Crop and Grassland Science of the Federal Agricultural Research Centre (FAL, Braunschweig). Designed specifically for routine use in genebanks, this method uses a very simple ultra-rapid freezing approach and was applied to a wide range of varieties within the Federal Centre for Breeding Research on Cultivated Plants (BAZ, Quedlinburg) Potato Collection.
   After several years storage in liquid nitrogen, shoot tips from a random sample of 51 varieties were thawed and the survival and shoot regeneration percentages compared to those measured immediately after freezing. There were no major changes in either survival or recovery of frozen apices. Data presented are not the outcome of a systematic experiment but from that accumulated during our work from 1992 to 1999. 

Keywords: potato, cryopreservation, cryostorage, recovery, droplet freezing, Solanum tuberosum.

CryoLetters 24, 43-48 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

DEVELOPMENT OF A CRYOPRESERVATION PROTOCOL FOR THE MICROCYCLIC RUST-FUNGUS Puccinia spegazzinii

 Matthew J. Ryan* & Carol A. Ellison

CABI Bioscience, Bakeham Lane, Egham, TW20 9TY, UK (m.ryan@cabi.org)

Abstract

The rust fungus Puccinia spegazzinii (Basidiomycotina: Uredinales) has been identified as a potential classical biological control agent for the invasive weed Mikania micrantha (Asteraceae). Long-term, live storage of this pathogen is required for reference. As biotrophs, almost all rusts species cannot be preserved by traditional cryopreservation protocols, which rely on in vitro culture techniques. In addition, the embedded teliospores and delicate basidiospores of this microcyclic rust are not amenable to direct plunge freezing. Continuous culture of the rust on living plants is both laborious and expensive, so a variety of approaches for cryopreservation and storage were tested. These methods included traditional approaches to fungal cryopreservation such as variation of cooling rate regime and alginate encapsulation techniques. However, an in situ cryopreservation technique was the only method identified as having any potential for the long-term cryopreservation of the 10 isolates tested. Material from either petiole or stem tissue remained viable after cryopreservation, determined by the ability of the material to produce basidiospores. However, despite great progress being made in developing an optimal cryopreservation method, infection of the host plant by basidiospores produced from previously cryopreserved teliospores, embedded in leaf petioles, was not achieved. 

Keywords: preservation, Basidiomycotina, biocontrol, plant pathogen

CryoLetters 24, 49-56 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ULTRASTRUCTURE OF CARP Cyprinus carpio SPERMATOZOA AFTER COOLING, DILUTION AND FREEZE-THAWING

S.I. Drokin^1*, the late H. Stein² and T.P. Govorukha¹

¹Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, 23 Pereyaslavskaya Str., Kharkov 31015, Ukraine. Email: sidrokin@rambler.ru
*To whom correspondence should be addressed.
² Technical University Munchen, Freising, Germany.

Abstract

Dilution of the carp spermatozoa with a cryoprotective medium was shown to inflict serious injuries to the fine structures of some spermatozoa. The amount of the injured spermatozoa increases after cryopreservation. Examination of the freeze-fracture micrographs of the heads of the carp spermatozoa, showed that their protoplasmic faces were covered with the particles in high concentrations. These particles do not form clusters after cooling the spermatozoa down to +5C and dilution with a cryoprotective medium. For the first time the authors observed the particles to form geometrically shaped regular formations on the outer membranes of the carp spermatozoa.
On the basis of the results obtained the authors propose that high susceptibility of the carp spermatozoa to osmotic exposures is apparently due to some particular features of the fine structures of their membranes and to their expected probably low heterogeneity.

Keywords: spermatozoa, freeze-thawing, fine structure, cryopreservation, membranes, fish

CryoLetters 24, 57-64 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF CARROT (Daucus carota L.) CELL SUSPENSIONS AND PROTOPLASTS BY VITRIFICATION

Yong Chen¹ and Jun-Hui Wang²*

¹Department of Biological and Environmental Sciences, Wenzhou Normal College, Wenzhou 325003, China
²College of Life Sciences, Zhejiang University, Hangzhou 310012, China (E-mail: junhuiwang@zju.edu.cn)

Abstract

Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 ˚C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25 % PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate - propidium iodide) staining) was achieved after cryopreservation.

Keywords: Daucus carota, vitrification, cryopreservation, cell suspension, protoplast

Abstracts

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