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Astracts: CryoLetters 20 (6), 1999

CryoLetters 20, 337-338 (1999)

Editorial: Flat Earthers of the world unite

Felix Franks



CryoLetters 20, 339-346 (1999)

Effect of osmotic stress on the dehydration tolerance of Solanum tuberosum shoot tips

M. Grospietsch*, E. Stodulková and J. Zámecnik

*Research Institute of Crop Production, Drnovská 507, Prague 6, CZ - 161 06, Czech Republic

Four different preculture treatments were tested for their ability to enhance survival of potato shoot tips (cv. Desirée) after both dehydration and cryopreservation. The encapsulation/dehydration method and rapid freezing were used. Further, accumulation of sucrose, glucose, fructose, glycerol and proline in the apices during the treatments was measured. Very high internal sucrose concentrations (up to 27.8 % of dry mass) was essential to reach high survival rates after cryopreservation, but not after dehydration only. The highest survival rate (78.8%) was obtained when osmotic stress was induced by adding 2 M sucrose solution to donor plants for 5 days, followed by 0.7 M sucrose preculture of isolated shoot tips for 1 day. The results demonstrate that drought hardening would be able to replace the effect of low temperatures in such plants, which are not able to be cold hardened.



CryoLetters 20, 347-352 (1999)

Cryopreservation of encapsulated sugarcane apices: Effect of storage temperature and storage duration

*M.T. Gonzalez-Arnáo, C. Urra, F. Engelmann, R. Ortiz and C. de la Fe

*Univ. de la Habana, Fac. de Biologia, Calle 25 e/ J e I, Vedado, C. Habana, Cuba (present address)

Apices sampled on in vitro plantlets of sugarcane were cryopreserved using the encapsulation-dehydration technique, comprising pregrowth for 24 h in liquid medium with 0.75 M sucrose, desiccation to 20-25 % moisture content (fresh weight basis) and rapid immersion in liquid nitrogen. Apices of one variety were stored for up to one year at -196° C, -70° C or -25° C, with or without previous transitory immersion in liquid nitrogen. Whereas the recovery percentage of apices stored at -196° C remained high and constant throughout the experiment, recovery of apices conserved at -70° C and -25° C decreased dramatically within 0.5 day, and was nil after 10 or 120 days in storage at -25° C and -70° C, respectively. The recovery percentage of apices of four additional varieties remained stable throughout a 1 year storage period at -196° C. No differences were noted for 6 agronomic traits between plants regenerated from control and cryopreserved apices of two sugarcane varieties.



CryoLetters 20, 353-362 (1999)

Impact of cryoprotective agent exposure on intracellular calcium in mouse oocytes at metaphase II.

B. Litkouhi, W. Winlow and *R. G. Gosden

*Dept Obstetrics and Gynaecology, McGill Univ., Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada (present address)

This is the first study to report the impact of cryoprotective agent (CPA) exposure on [Ca+2]i in oocytes. [Ca+2]i was measured in mature unfertilised mouse oocytes after step changes in membrane-permeable CPAs (1.5 M dimethyl sulfoxide, 1.5 M 1,2-propanediol, 1.5 M glycerol) and non-penetrating CPA (0.1 M sucrose) at room temperature using fluorescence microscopy and the Ca+2 sensitive dye fura-2 AM. Oocytes were classified as young or aged according to the time of collection and responses were compared to controls exposed to isotonic oocyte preservation medium only. Results show that in young oocytes only immersion in 1.5 M propanediol consistently increased [Ca+2]i. However, there may also be more subtle factors, such s oocyte age, normal population variation and temperature, affecting the response and subsequent viability of oocytes after CPA exposure.



CryoLetters 20, 363-370 (1999)

Effects of ingestion and excretion of ice-nucleating-active bacteria on the supercooling ability and cold hardiness in larvae of the clover leaf weevil Hypera punctata

M. Watanabe

Dept. Insect Physiology and Behavior, National Institute of Sericulture and Entomological Science, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan

Ingesting of ice-nucleating-active (INA) bacteria Pseudomonas syringae or Xanthomonas campestris decreased the supercooling ability and thereby the cold hardiness in 4th instar larvae of Hypera punctata. Seventy percent of the larvae that had ingested a large number of live P. syringae died from freezing at -5ºC, while larvae that had ingested X. campestris did not die at the same temperature. There was a close positive correlation between the increase in the larval supercooling point (SCP) and the number of live P. syringae taken into the gut, suggesting that INA bacteria in the gut themselves increase the larval SCP. Most of the ingested P. syringae were excreted from the larval gut within 5 days at 25ºC, resulting in the decrease of the larval SCP. These results indicate the potential use of P. syringae for the biological control in larvae of this beetle which continue to feed during winter and are sometimes exposed to subzero temperatures around -5ºC at the overwintering site.



CryoLetters 20, 371-376 (1999)

Extended cold acclimation and recovery medium alteration improve regrowth of Rubus shoot tips following cryopreservation

Y. Chang and *B. M. Reed

*USDA/ARS, National Clonal Germplasm Repository, 333447 Peoria Rd, Corvallis, OR 97333, USA.

Extended cold acclimation (CA) of shoot cultures in alternating low temperatures [22ºC, 8 h light; 1ºC, 16 h dark] improved the recovery of cryopreserved shoot tips. As the duration of CA prior to cryopreservation increased from 1 to 3 weeks, Rubus parvifolius L. shoot tip survival increased from 63 to 90 % and shoot formation increased from 25 to 75 %. Six to ten weeks of CA were required to achieve high survival and shoot formation in R. caesius L. and improve shoot survival from 8 to 70-80 % and shoot formation from zero to 60-80 %. Eliminating indole-3-butyric acid from the recovery medium decreased callus formation and increased direct shoot formation for both species. Histological studies showed that R. parvifolius shoot tips continued to grow and regenerated directly from the meristematic domes following liquid nitrogen exposure. The upper axillary buds often survived and regrew along with the apex. No shoots regenerated from callus produced on margins of leaf primordia and damaged domes.



CryoLetters 20, 377-382 (1999)

Effect of cryoprotectant treatment and post-thaw washing on the survival of cultured rice (Oryza sativa L.) cells after cryopreservation

*K. Watanabe, A. Kuriyama, F. Kawai and M. Kanamori

*Dept. Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan

Cultured rice cells differing in cryoprotectability were loaded with 5 % dimethyl sulfoxide and 10 % D-glucose at 0ºC for 1 h. Both the sugar content and osmotic pressure of loaded cells became higher than those of untreated cells. In highly cryoprotectable cells, those values were reduced to the level of untreated cells by washing with liquid medium. For the poorly cryoprotectable cells, however, they remained higher than those of the untreated control cells after the washing. Two-dimensional electrophoresis of proteins extracted from the cells during post-thaw culture showed that cell content were lost from the poorly cryoprotectable cells at the initial stage of post-thaw culture. These results suggest that frozen-thawed, poorly cryoprotectable cells are damaged more or less by osmotic stress and do not survive after cryopreservation in liquid nitrogen.



CryoLetters 20, 383-392 (1999)

Characterisation of rapid cold-hardening response in the overwintering mature larvae of pine needle gall midge Thecodiplosis japonensis

*Y. Li., H. Gong and H-Y. Park

*State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, CAS, Beijing 100080, China.

The pine needle gall midge, Thecodiplosis japonensis Uchida et Inouye, overwinters as a third-instar mature larva in the litter layer or soil surface in Korea. The supercooling point (SCP) of the larvae remains constant at -20.6 ± 2.3ºC during overwintering. No larva survives below the SCPs and overwintering larvae adopt a freeze avoiding strategy for survival of subzero temperatures. A rapid cold hardening response is found in field-sampled T. japonensis larvae from the end of December to the end of February in 1997/98. The acquired cold hardiness is transient and is rapidly lost (after 15 min at 27ºC). Rapid cold hardening process is more effective in maintaining larval survival than long term hardening process. It indicates that different mechanisms exist in the rapid and long-term cold hardening responses in overwintering T. japonensis larvae.



CryoLetters 20, 393-404 (1999)

Low temperature induced cryoprotectant synthesis by the infective juveniles of Steinernema carpocapsae: biological significance and mechanisms involved

*L. Qiu and R. Bedding

*CSIRO Entomology, P Box 1700, ACT 2601, Australia

The infective juveniles (IJs) of Steinernema carpocapsae synthesised trehalose but not glycerol at low temperatures. Equilibrium trehalose levels were temperature dependent. When the IJs were incubated aerobically in tap water at temperatures ranging from 2 to 14ºC, their trehalose levels increased from 1.9n % dry weight to equilibrium levels ranging from 3.4 % at 14ºC to 6 % at 5-8ºC. Noticeable increases in trehalose levels in the IJs of four other species of entomopathogenic nematodes (ENs) exposed to 5ºC indicated that this is likely to be a common characteristic of the IJs of ENs. Cold induced IJs had a much higher tolerance to severe osmotic dehydration. When aging IJs, which have lower energy reserves than fresh ones, were exposed in the same way to 5ºC for 7 days, their trehalose levels were lower than those of fresh IJs but the survival rates of the IJs did not drop substantially. Changes in lipid, glycogen and protein levels of IJs during cold induction and subsequent recovery indicated that trehalose was not synthesised from glycogen, but from lipids and/or proteins. The processes involved in low temperature induced cryoprotectant synthesis by ENs are discussed and compared to those found in insects.

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