Abstracts: CryoLetters 24 (2), 2003

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

CryoLetters 24, 65 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK




CryoLetters 24, 67-76 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


V.I. Grischenko1, A.V. Dunaevskaya1 and V.I. Babenko2

1Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, 23 Pereyaslavskaya str., Kharkov 61015, Ukraine
2Institute of Low Temperature Physics and Engineering, 47 Lenin’s Ave, Kharkov, 61103, Ukraine


The effect of cryopreservation on human spermatozoa has been investigated during the past few decades. The majority of current cryopreservation protocols are carried-out using low cooling rates. However, theoretical calculations have shown that for human spermatozoa an optimal cooling rate is about 7x103 ºC/min. In our work we have studied the effect of cryopreservation with high cooling rates, variation of osmolarity of the cryoprotectant medium and the glycerol content. The results of experiments have demonstrated that within the range of high cooling rates, after thawing the dependency of sperm survival on the cooling rate has a maximum recovery at 2.5-3.3 x103 ºC/min in moderately hyperosmolar medium, containing 4-5% glycerol. When decreasing the cooling rate down to 1.75-2.5 x103 ºC/min there was a statistically significant reduction in sperm motility. Using the cooling rate of 8-11 x103 ºC  /min only a small percentage of spermatozoa retained their motility.

Keywords: cryopreservation, human spermatozoa, cooling rate, glycerol



CryoLetters 24, 77-84 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Genetic stability Assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera L. using rapd, biochemical and Morphological Analysis

Sonali Dixit1, B. B. Mandal1*, Sangeeta Ahuja1, 2 and P. S. Srivastava3

1Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, India.
2Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia-22908, USA (current address).
3Centre for Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India.
*To whom correspondence should be addressed. E-mail:


Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.

Keywords: RAPD, genetic stability, biochemical analysis, cryopreservation, yams.



CryoLetters 24, 85-94 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


M.T. González-Arnao1, J. Juárez, C. Ortega, L. Navarro and N. Duran-Vila*

Departamento de Protección Vegetal y Biotecnología. Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada (Valencia), Spain. (E-mail:
1On leave from Facultad de Biología, Universidad de La Habana, Calle 25 e/ J e I, Vedado, C. de La Habana, Cuba. (E-mail: and


Cryopreservation of ovules and somatic embryos from several genotypes of citrus was achieved using the encapsulation-dehydration technique. Survival of cryopreserved ovules was occasional and erratic after different pregrowth conditions in liquid medium with 0.75M, 1M or up to 1.25M sucrose. An efficient cryopreservation protocol was established for somatic embryos derived from two embryogenic sources (ovules and cut thin layer explants from stigma, style and ovaries). High survival rates (75-100%) were consistently obtained after 1 day pregrowth in 0.75M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in liquid nitrogen. The histological study showed that embryos subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation.  

Keywords: Germplasm, cryopreservation, citrus, ovules, somatic embryos, encapsulation-dehydration.



CryoLetters 24, 95-102 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Barry J Fuller

University Department of Surgery, Royal Free & University College Medical School, London NW3 2QG, UK.


This review was undertaken to identify responses of mammalian cells to cold temperatures, such as might be encountered in therapeutic procedures where body temperatures are lowered, in preservation of cells and organs, and in cryopreservation. In general, cold elicits a range of stress responses through identified signaling pathways, which may determine the survival or otherwise of the cells. Under conditions of mild hypothermia, there is evidence for responses which reflect an ordered acclimation to the new environment, whilst deep cooling invokes a more generalised stress response.

Keywords: low temperatures; hypothermia; cryopreservation; gene products; stress response.

The concepts reported here were presented to the Society for Low Temperature Biology symposium (2002, London) entitled Chromosomes, Genes and Cryobiology.



CryoLetters 24, 103-110 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


R.F.Gagliardi1, G.P.Pacheco1, L.A.Carneiro1, J.F.M.Valls2, M.L.C. Vieira3, and E.Mansur1*

1Laboratório de Micropropagação e Transformação de Plantas, Universidade do Estado do Rio de Janeiro, Brazil;
2EMBRAPA/CENARGEN, Brasília, DF, Brazil;
3Depto de Genética ESALQ – Universidade de São Paulo; *author for correspondence (e-mail: )


A storage protocol at cryogenic temperature was established for shoot apices from in vitro plants of the cultivated groundnut (Arachis hypogaea) and wild Arachis species (A. retusa and A. burchellii) using a basic vitrification protocol with direct immersion in liquid nitrogen (LN). The effect of pre-treatments of donor-plants with ABA as well as of different supplements in the post-thaw culture medium was studied. After rapid warming at 40°C, the explants were cultured on MS medium devoid of growth regulators (MS0) or MS supplemented with 4.4M benzylaminopurine (BAP) and 0.5M naphthalene acetic acid (NAA) plus 5M silver nitrate (AgNO3), 0.25% polyvinylpyrrolidone (PVP) or 0.2% activated charcoal. Non-frozen explants from the three species formed one shoot through meristematic amplification when cultured on MS0 medium. These explants also developed callus on MS supplemented with growth regulators (4.4M BAP and 0.5M NAA) alone or plus PVP or AgNO3. Callus formation was suppressed in the presence of activated charcoal. Post-thaw regeneration ocurred only through indirect organogenesis on media containing AgNO3 or PVP. Preculturing on medium supplemented with abscisic acid (ABA) improved regrowth rate in these media. Recovery failed to occur in the presence of activated charcoal. The genetic stability of shoots of A. burchellii originated from shoot apices was analyzed through Random Amplified Polymorphic DNA (RAPD) markers.

Keywords: germplasm preservation, groundnut, vitrification, cryopreservation, genetic stability, RAPD



CryoLetters 24, 111-118 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cold resistance in the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera : Tenebrionidae)

C. Salin1*, P.Vernon1 and G.Vannier2

1 UMR 6553 CNRS, Université de Rennes I, Station Biologique, 35380 Paimpont, France
2 UMR 8571 CNRS, Muséum national d'histoire naturelle, Laboratoire d'écologie générale, 4 avenue du Petit-Château, 91800 Brunoy, France.
*Current address: Laboratoire d’Ecologie et de Biogéographie, Centre de Recherche sur la Biodiversité, Université catholique de Louvain, place Croix du Sud 5, 1348 Louvain la Neuve, Belgique. E-mail:

  • Abstract
  • We have investigated cold resistance, measured by the supercooling point (SCP) temperature, in life stages of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae), collected in Brittany poultry houses. Mean SCP values drastically increased during the insect ontogeny: egg (-26.1°C), first instar larvae (-21.6°C), last instar larvae (-15.5°C), pupae (-11.6°C), teneral adults (-12.0°C) and mature adults (-13.1°C). Nymphal metamorphosis and adult maturation did not promote substantial decrease of freezing resistance. The SCP values reflect the physiological states of the developmental stages especially the absence of ice nucleating agents: (i) lower SCP values in egg and unfed newly-emerged larvae I (i.e. -25.1°C), (ii) higher SCP values in fed larvae (i.e. -14.7°C), pupae and adults most likely due to the presence of ice nucleation sites in the gut. A tropical species, A. diaperinus, seems not to use its potential cold hardiness even in winter to remain in this warm habitat in temperate regions.

    Keywords: cold hardiness, supercooling point, ontogenesis, Coleoptera, Alphitobius diaperinus.



    CryoLetters 24, 119-124 (2003)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


    R.K. Tyagi* and T. Hymowitz

    Department of Crop Sciences, University of Illinois, 1102 South Goodwin Avenue, Urbana IL 61801, USA
    *Corresponding author, Present address: National Bureau of Plant Genetic Resources, New Delhi-110 012, India . E-mail:


    Pollen of 12 genotypes of the annual soybean and its wild perennial relatives were stored without pre-desiccation at low temperatures (-20ºC and -196ºC) and tested for their viability in vitro. The influence of cryopreserved pollen on pod set and seed production was also investigated. Cryopreserved pollen of all the genotypes showed germination in vitro. Pollen of annual soybean stored at -20ºC retained their viability for 4 months, however, pollen of its wild perennial relatives at same storage conditions failed to germinate in vitro. Flowers pollinated with cryopreserved pollen had similar pod set and number of seeds/pod as those pollinated with fresh pollen. Results of this study suggest that cryopreservation of pollen can be used successfully for soybean breeding, and also offers the possibility of conserving the haploid gene pool of soybean and wild perennial species in a cryobank facility.

    Key words: Cryopreservation, hybridization, germplasm conservation, Glycine spp., soybean



    CryoLetters 24, 125-132 (2003)
    © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


    A. Rajamohan1, R.A. Leopold2*, W.B. Wang3, M. Harris1, S.D. McCombs4, N. C. Peabody4 and K. Fisher4

    1Entomology Department, North Dakota State University, Fargo, ND 58105, USA
     2UDSA/ARS Biosciences Research Laboratory, Fargo, ND 58105, USA
    3Via-Cell, Worcester, MA 01605, USA
    4USDA/APHIS Plant Protection Laboratory, Waimanalo, HI 96795, USA


    In this paper we present a procedure to cryopreserve the embryos of a tephritid, the Mediterranean fruit fly (Ceratitis capitata), by vitrification.  Developmental stages between 24 and 32 hours after oviposition were examined for tolerance to cryopreservation. Embryos, 27-hr-old and incubated at 29°C, were found to be at the most suitable stage for treatment. Effects of the previtrification steps of our protocol, dechorionation, permeabilization, cryoprotectant loading, and dehydration, on survival to hatching were also assessed. Dechorionation did not affect viability, while isopropanol and a hexane treatment used in the permeabilization step of the protocol reduced hatching by about 15%.  This reduction was dependant on the amount of isopropyl alcohol carried over into the hexane rinse. The remaining previtrification steps reduced hatching by an additional 10%.  After optimization of the procedure, normalized hatching was 44% after vitrification in liquid nitrogen vapor followed by storage under liquid nitrogen for a test period of 7 days. Post cryopreservation larval diets containing wheat bran, corncob grits, or agar as the base were examined for survival to pupation and emergence.  A yield of 34% egg to adult emergence was obtained when the agar-based diet was used for rearing larvae that had experienced cryopreservation during the embryonic stage.

    Keywords: vitrification, Tephritidae, Ceratitis capitata, medfly


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