Abstracts: CryoLetters 21 (6), 2000

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

CryoLetters 21, 327-332 (2000)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Peter Convey* & M. Roger Worland

British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK


Free-living microbivorous nematodes become numerically dominant in Antarctic terrestrial faunas as environmental conditions become more severe, while also reaching very high levels of abundance in moist, vegetated habitats.  Nematodes have little resistance to freezing via exogenous ice nucleation, such as would occur as their microhabitat freezes.  We report the results of experiments testing the ability of seven maritime Antarctic nematode taxa to survive freezing in small water droplets at high sub-zero temperatures. Isolated individuals of these species possessed supercooling characteristics similar to those previously reported (supercooling points -6 to -25C).  When frozen in water at -3 to -6C, most showed high (> 70%) survival both (i) after rapid cooling (1C min-1) to c. -60C followed by immediate rewarming, and (ii) when held for 7-12 h at either -10 or -30C,  although the proportions surviving varied between species. We propose that the ability to survive freezing while fully hydrated at high sub-zero temperatures is one of the most important aspects of these species’ survival tactics.

Keywords: Antarctic, nematode, freeze tolerance, survival



CryoLetters 21, 333-338 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Peter Convey* & M. Roger Worland

British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 0ET, UK


In studies of three common, freezing susceptible, Antarctic microarthropods, the springtail Cryptopygus antarcticus and the mites Alaskozetes antarcticus and Halozetes belgicae, we report (i) the consequences on cold tolerance of cooling in contact with water, and (ii) the risk of freezing when held at temperatures above the typical freezing point (measured using standard techniques) for up to 12 h. The springtail showed no change in SCP distribution when in contact with freezing water while, in contrast, the mites showed clear shifts towards decreased cold tolerance, in addition to death of c. 33% of individuals during the freezing of the water.  The springtail showed a bimodal SCP distribution, with the population divided into “high”(typically -8 to -12C) and “low” (typically below -20C) groups.  Some animals held at temperatures above these values froze, over a timescale between minutes and several hours. These results highlight the danger of equating standard cold tolerance measures with mortality risk under more realistic water and thermal regimes.

Keywords: Antarctic, mite, springtail, exogenous nucleation, mortality risk



CryoLetters 21, 339-348 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU



Oldřich Nedvĕd

Institute of Entomology, Academy of Sciences of the Czech Republic,  Faculty of Biological Sciences, University of South Bohemia,  Branišovská 31, 370 05 České Budějovice, Czech Republic


Two main cold hardiness strategies of insects – freeze tolerance in some species, and overwintering in a supercooled state without tolerance of freezing in many others – were recently reclassified. However, I present several problems with the current systems. My suggested classification is based on clearer definitions of the causes of cold injury. I recognize three main mortality factors: freezing of body liquids, cold shock, and cumulative chill injury. Presence or absence of each of these factors produce eight combinations. I have named the eight classes after Snow White and the Seven Dwarfs to avoid nomenclatural confusion. Some of these classes are probably not used as tactics against cold injury by any insect species. Other classes contain so many species that they might be reclassified in more detail, using values of supercooling point and other quantitative parameters. However, widely comparable parameters, like the upper limit of cold injury zone and the sum of injurious temperatures are still rarely published, thus we still lack comprehensive data for multivariate analyses. Every cold hardiness strategy should be characterized by a meaningful class or subclass together with the physiological, biochemical, and behavioural mechanisms employed by the insects. I also point out the existence of strategies that combine two tactics – either a switching strategy (during preparation for winter, population “chooses” which tactic will be used), or a dual strategy (individuals are ready to use one of the tactics depending on the prevailing environmental conditions).

Keywords: cold hardiness strategy, cold tolerance, freeze tolerance, chill tolerance, supercooling point, lower lethal temperature



CryoLetters 21, 349-356 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


T. Niino1, A. Hettiarachchi2, J. Takahashi2and P. K. Samarajeewa2*

1Dept. of Upland Farming, Tohoku National Agricultural Experiment Station, Arai, Fukushima 960-2156 Japan
2Plant Genetic Resources Center, Gannoruwa, Peradeniya, P.O. Box 59, Sri Lanka.
*to whom correspondence should be addressed.


We succeeded in cryopreserving of innala (Solenostemon rotundifolius) in vitro-grown young lateral buds by vitrification. Nodal segments from in vitro-grown shoots (2-4 mm in length) were cultured on MS medium containing 0.1M sucrose in Petri dishes for 3 weeks under 16-h photoperiod at 25C. This pre-growth induced a large number of uniform young lateral buds. Nodal segments (0.5 to 1.0 mm in length) with two lateral buds were dissected from the shoots and precultured with 0.3 M sucrose for 2 days at 25C.  They were then treated with loading solution containing 2 M glycerol and 0.4 M sucrose (LS solution) for 20 min at 25C and dehydrated with the PVS2 vitrification solution for 18 min at 25C prior to either rapid immersion in liquid nitrogen. Surviving lateral buds resumed growth within 3 days and developed shoots without intermediary callus formation. The average growth recovery after cryopreservation amounted to 85%.

Keywords: Innala (Solemostemon rotundifolius), cryopreservation, vitrification, lateral bud.



CryoLetters 21, 357-366 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Rida A. Shibli1 * and Karim H. Al-Juboory2

1 Biotechnology Center - Agriculture, Jordan University of Science and Technology, Irbid, Jordan
2 Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61801, USA
* Corresponding author, Fax 962-2-7095123, email:


Olive (Olea europea L.) somatic embryos were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification. In the encapsulation-dehydration procedure, a maximum of 48% embryo survival  was obtained when bead moisture content was decreased to 21.1% after 4 h dehydration. Preculture of embryos for 4 d in medium containing 0.75 to 1.25 M sucrose produced higher (40 to 34 %, respectively) regrowth after cryopreservation using encapsulation-dehydration procedure. Dehydration of beads for 3 h in PVS2 ensured higher survival (64%) of encapsulated-vitrified and cryopreserved (EVN) somatic embryos. Thermal treatment of embryogenic callus for 1 d at 30şC was very effective to increase survival of encapsulated-dehydrated and cryopreserved (EDN) (58%) and EVN (68%) embryos. Plantlets produced from control and cryopreserved embryos were phenotypically similar.

Keywords: Cryopreservation; olive; Olea europea L.; encapsulation-dehydration; encapsulation-vitrification; somatic embryos.



CryoLetters 21, 367-378 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


Dominique Dumet1#, William Block2, Roger Worland2, Barbara M.Reed3 and Erica E Benson*1

1Plant Conservation Group, School of Science and Engineering, University of Abertay Dundee, Dundee, Bell Street, Scotland, DD1 1HG
2British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 OET, UK;
3USDA-ARS, National Clonal Germplasm Repository, 33447, Peoria Road, Corvallis, Oregon, OR 97333, USA.
#Present address: Schools of Life and Environmental Sciences, University of Natal, Durban, 4041 South Africa.
*Author to whom correspondence should be addressed.


DSC analysis was performed at three points in the cryopreservation process on encapsulated-dehydrated meristems of Ribes ciliatum. Meristems were excised from shoots pre-treated with either sucrose or glucose, encapsulated in alginate beads, dehydrated in sucrose solutions, air dried, and plunged in liquid nitrogen.  Thermal analysis revealed glass transitions during cooling of air-desiccated meristems, however, on rewarming a small endothermic event was detected suggesting glass destabilization can occur. Interestingly, this did not occur in alginate beads or meristems when these components were cooled and rewarmed separately.  The possibility exists that thermal and moisture gradients may arise within the alginate bead/tissue complex and we propose that the heterogeneous composition of the meristems and the surrounding alginate may promote ice nucleation on rewarming. The significance of this regarding the stabilization of glasses formed in alginate beads and their encapsulated meristems is discussed. This study also reports an approach to Ribes cryopreservation in which the pregrowth of shoots in 0.75M sucrose for 1 week can be used as a substitute for cold acclimation.


Keywords: Cryopreservation, water content, differential scanning calorimetry, desiccation, thermal analysis, vitrification, Ribes



CryoLetters 21, 379-388 (2000)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU


S.R Turner 1,2,*,  B. Tan2 , T. Senaratna1 , E. Bunn1 , K. W. Dixon1 , D.H. Touchell3

1 Kings Park and Botanic Garden, West Perth, WA, 6005, Australia.
2 Curtin University of Technology, Bentley, WA, 6102, Australia.
3 USDA-ARS National Seed Storage Laboratory, 1111 South Mason St, Fort Collins,             Colorado, 80521, USA.
*  Corresponding author; email:


Somatic embryos were used to develop a cryopreservation protocol for Macropidia fuliginosa, a commercially-important species endemic to the south-west of Western Australia. Somatic embryos were allowed to develop from embryogenic callus for three weeks on an kinetin medium prior to processing. These were transferred and cultured on a agar solidified basal medium supplemented with 0 to 0.6 M sorbitol for 2 d prior to incubation in Plant Vitrification Solution Two (PVS2). Following this, embryos were then washed in 1 M sucrose solution (treated controls) or cooled in liquid nitrogen (LN). Cooled embryos were then warmed and washed in sucrose solution. Highest survival for cooled treatments (67.3%) was achieved by preculture with 0.4 M sorbitol, then incubation in PVS2. Further experimentation varying pre-culture duration (2 or 3 d) and incubation on either glycerol (0.8 M) or sorbitol (0.4 M) indicated that very high survival (90.6%) of embryos was achievable by adopting a 2 d preculture period on 0.8 M glycerol. The phenotype and growth rates of plants obtained using this protocol were similar to those  of parent plants. This optimised procedure was then applied to tissue culture-derived shoot apices of the same clone also resulting in a high survival rate (84.4%).

Keywords: Macropidia fuliginosa, PVS2, sorbitol, glycerol, somatic embryos

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