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Abstracts: CryoLetters 30 (1), 2009

 

 

Volume 30, No. 1 January/February

ISSN 0143-2044

 

 


MITOCHONDRIAL FUNCTION AFTER LIVER PRESERVATION IN HIGH OR LOW IONIC-STRENGHT SOLUTIONS: A COMPARISON BETWEEN UW-BASED AND SUCROSE-BASED (SBS) SOLUTION
Alexander Yu. Somov, Olga A. Semenchenko, Colin J. Green, Alexander Yu.  Petrenko and Barry J. Fuller

1-12

 

 


A SIMPLE ASSAY SYSTEM TO MONITOR THE POTENTIAL FOR CONTAMINATION DURING DIFFERENT STAGES OF CRYOPRESERVATION
G. J. Morris

13-18

 

 


OF PLANTLET NODES OF Dioscorea opposita THUNB. USING A VITRIFICATION METHOD
Mingjun Li, Xiting Zhao, Senrong Hong, Xiaoli Zhang, Ping Li, Jun Liu and Conghua Xie

19-28

 

 


CRITICAL THERMAL MINIMA, THEIR SPATIAL AND TEMPORAL VARIATION AND RESPONSE TO HARDENING IN Myrmica ANTS
Andrey Maysov and Vladilen E. Kipyatkov

29-40

 

 


RECOVERY OF PLANTS FROM PEA AND STRAWBERRY MERISTEMS CRYOPRESERVED FOR 28 YEARS
Karen L. Caswell and Kutty K. Kartha

41-46

 

 


COLD ACCLIMATION IMPROVES REGROWTH OF CRYOPRESERVED APPLE SHOOT TIPS
Svetlana V. Kushnarenko, Natalia V. Romadanova, and Barbara M. Reed

47-54

 

 


CRYOPRESERVATION OF EMBRYOGENIC CULTURES OF 'BREWSTER' LITCHI (Litchi chinensis SONN.) AND ITS EFFECT ON HYPERHYDRIC EMBRYOGENIC CULTURES
Guillermo Padilla, Pamela Moon, Irene Perea and Richard E. Litz

55-63

 

 


CRYOPRESERVATION OF EMBRYOGENIC CALLUS OF Dioscorea bulbifera BY VITRIFICATION
Senrong Hong, Minghua Yin, Xinghua Shao, Aiping Wang and Weihong Xu

64-75

 

 


Society for Low Temperature Biology Meetings Abstracts
University of Copenhagen, Denmark 2008

76-88

 

 

 

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CryoLetters 30 (1), 1-12 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

MITOCHONDRIAL FUNCTION AFTER LIVER PRESERVATION IN HIGH OR LOW IONIC-STRENGHT SOLUTIONS: A COMPARISON BETWEEN UW-BASED AND SUCROSE-BASED (SBS) SOLUTION

Alexander Yu. Somov1, Olga A. Semenchenko1, Colin J. Green3, Alexander Yu.  Petrenko1 and Barry J. Fuller2

1Institute for Problems of Cryobiology and Cryomedicine, Kharkov, Ukraine,
2University Department of Surgery and Liver Transplant Unit, Royal Free Hospital and School of Medicine, London, United Kingdom
3Department of Surgical Research, Northwick Park Institute for Medical Research, United Kingdom
*Correspondence author e-mail:
somov@ukr.net

Abstract

In this study we evaluated mitochondrial function after liver cold storage and normothermic reperfusion. The preservation solutions were: modified University of Wisconsin (mod UW); sucrose-based solution (SBS). After cold preservation liver was re-perfused for 1 hour in vitro with Krebs-Ringer buffer at 37C. Samples of tissue were taken for ATP determination. Mitochondrial respiratory parameters, succinate oxidase complex activity, mitochondrial H+-ATPase and intramitochondrial potassium concentration were assayed. It was shown, that brief (1h) cold storage and subsequent normothermic reperfusion revealed no difference in liver ATP content between mod UW and SBS groups but resulted in a gradual decrease of 50% after 24hour storage and reperfusion. Mitochondrial potassium ion concentration increased by 40% after 1-hour cold storage in the mod UW as compared to control (P < 0.05) and SBS. After brief cold storage ADP and uncoupler-stimulated respiration increased by 120% in SBS group, unlike mod UW, when succinate was used as substrate, and was more pronounced after 24 h. Succinate oxidase complex activity did not change over either cold storage or warm reperfusion. Mitochondrial H+-ATPase activities in SBS and mod UW did not differ and both were inhibited after 24-hour cold storage. Our data demonstrate that low ionic strength preservation solution can substantially modulate mitochondrial energy turnover due to substrate oxidation increase. Many of the changes in mitochondrial function follow brief exposure to low temperatures.

Keywords: liver cold storage, University Wisconsin solution, sucrose-based solution, mitochondrial respiratory complexes, oxygen consumption, succinate oxidase, intramitochondrial potassium.

 

 

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CryoLetters 30 (1), 13-18 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

A SIMPLE ASSAY SYSTEM TO MONITOR THE POTENTIAL FOR CONTAMINATION DURING DIFFERENT STAGES OF CRYOPRESERVATION

G. J. Morris*

Asymptote Ltd., St John's Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK
* Correspondence author email:
jmorris@asymptote.co.uk

Abstract

A simple assay to monitor the potential for contamination during different steps of cryopreservation is described. The assay is based on the contamination of liquid nitrogen using crystals of sucrose hemi-heptahydrate, these are stable in liquid nitrogen, nitrogen vapour and ambient air and can be monitored by a simple assay which allows contamination risks to be evaluated in a direct, rapid manner.

Keywords: Cryopreservation, Contamination, Sucrose hemi-heptahydrate

 

 

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CryoLetters 30 (1), 19-28 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

OF PLANTLET NODES OF Dioscorea opposita THUNB. USING A VITRIFICATION METHOD

Mingjun Li1*,2, Xiting Zhao2, Senrong Hong2, Xiaoli Zhang2, Ping Li2, Jun Liu1 and Conghua Xie1*

1Key Laboratory of Horticultural Plant Biology of the Ministry of Education,  Huazhong Agricultural University, Wuhan 430070, China.
2College of Life Sciences, Henan Normal University, Xinxiang 453007, China. *Correspondence author e-mail:
xiech@mail.hzau.edu.cn

Abstract

A cryopreservation method by vitrification was developed for long-term storage of Dioscorea opposita Thunb., a valuable native medicinal plant species in Henan Province of China. The cryopreservation protocol was established with cultivar B and evaluated with another four cultivars, Tiegun, 47, Taigu and Huaiqing 1. The results showed that nodes with a bud excised from 60 d plantlets were desirable for the cryopreservation. The optimum procedure was established as: 1) the plantlets were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg L-1 KT and 0.02 mg L-1 NAA at 4C for 7 d before nodes with length of 1-1.5 cm were excised; 2) the nodes were precultured at 4C for 7 d on the MS supplemented with 10% dimethyl sulfoxide (DMSO) followed by loading with 60% Dioscorea vitrification solution 1 (DVS1): 22% (w/v) glycerol + 13% (w/v) ethylene glycol + 13% (w/v) polyethylene glycol + 10% (w/v) DMSO for 60 min at 0C and dehydrated with 100% DVS1 for 60 min at 0C; 3) the nodes were then immersed into liquid nitrogen (LN) directly and conserved for 180 d; 4) after rapid thawing in a water-bath at 37C, the nodes were rinsed four times with MS medium supplemented with 5% sucrose, then transferred to the MS medium supplemented with 2 mg L-1 kinetin (KT) and 0.02 mg L-1 NAA for regeneration. In the present research the regeneration rate of cv. B was about 77.1%, those of cvs. Tiegun and Huaiqing 1 were 67.2% and 54.0% respectively, while cvs. Taigu and 47 were about 40%. There were no visual changes observed between the plantlets regenerated from nodes with and without cryopreservation in terms of the morphology indices, indicating that the method established could be applicable to D. opposita with optimized protocol.

Keywords: Dioscorea opposita Thunb., Germplasm resources, Cryopreservation, Vitrification, Regeneration

 

 

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CryoLetters 30 (1), 29-40 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

CRITICAL THERMAL MINIMA, THEIR SPATIAL AND TEMPORAL VARIATION AND RESPONSE TO HARDENING IN Myrmica ANTS

Andrey Maysov* and Vladilen E. Kipyatkov

Department of Entomology, Faculty of Biology and Soil Science, St. Petersburg State University, Universitetskaya emb. 7/9, St. Petersburg, 199034, Russia.
*Correspondence author e-mail:
andrey.maysov@gmail.com

Abstract

In a changing environment the ability to adjust physiological functions to new conditions might be especially valuable for long-living ectothermic animals such as ants. With a simple method for estimating critical thermal minima of Myrmica ants we assessed variation of the minima and their response to lowered temperature. At a cooling rate of about 1C per minute the ants first displayed knock-down (at temperatures 1.5 to −0.2C) and only later immobilized completely (at −1.3 to −3.1C). Pre-chilling of ants at 5С for 1 h lowered the parameters significantly but not greatly (about 0.9С). Constant laboratory conditions (20С) raised the knockdown temperature and tended to lower the temperature of immobilization. In the same manner the two parameters changed in field conditions in June, but no significant change occurred through August and until the end of study season in mid-September. When populations from different geographic localities were compared, populations from the north showed lower knock-down temperatures. The magnitude of these apparently genetic differences among populations was comparable to the magnitude of plastic changes that occurred either naturally, or in experiments, or in laboratory culture. The little plasticity and low geographic variation of the critical thermal minima may indicate that the ability of the ants to withstand cold events and populate varying climates bases mainly on the protective properties of nesting substrate.

Keywords: chill coma, acclimation, hardening, plastic response, geographic variation, climate

 

 

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CryoLetters 30 (1), 41-46 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

RECOVERY OF PLANTS FROM PEA AND STRAWBERRY MERISTEMS CRYOPRESERVED FOR 28 YEARS

Karen L. Caswell* and Kutty K. Kartha

National Research Council of Canada / Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9. NRCC#50103 *Correspondence author e-mail: Karen.Caswell@nrc-cnrc.gc.ca

Abstract

Meristems isolated from in vitro strawberry shoot cultures (Fragaria x ananassa Duch. cv. Redcoat) and in vitro germinated pea seedlings (Pisum sativum L. cv. Century) were treated with cryoprotectants, slowly frozen to -40C (0.6C min-1 for pea and 0.84C min-1 for strawberry), and stored in liquid nitrogen in 1979. In 2007, several ampoules containing cryopreserved meristems of each species were thawed, cultured in vitro, and scored for viability. Meristems which differentiated into plants upon thawing and reculture were considered viable. Fifty-nine percent of the 91 strawberry meristems thawed in 2007 were viable after 28 years of cryopreservation, as compared to 56% viability after 8 weeks cryopreservation, as reported in the original study. Fourteen percent of the 78 pea meristems thawed in 2007 were viable after 28 years, as compared to 61% after 26 weeks of cryopreservation, as reported in the original study. With both strawberry and pea meristems there was variability in viability between ampoules. Viability between thawed ampoules of strawberry meristems ranged from 35 to 80%. Four of eight thawed ampoules of pea meristems contained no viable meristems, while the highest viability from a single ampoule was 77%. Shoots obtained from surviving meristems were rooted in vitro, transferred to soil, and produced phenotypically normal plants.

Keywords: Cryopreservation, long term storage, meristem, strawberry, pea

 

 

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CryoLetters 30 (1), 47-54 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

COLD ACCLIMATION IMPROVES REGROWTH OF CRYOPRESERVED APPLE SHOOT TIPS

Svetlana V. Kushnarenko1*, Natalia V. Romadanova1, and Barbara M. Reed2

1Institute of Plant Biology and Biotechnology at National Center of Biotechnology, Timiryazev str. 45, 050040, Almaty, Republic of Kazakhstan
2United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333 *Correspondence author e-mail:
svetlana_bio@mail.ru

Abstract

Cryopreservation is important for safeguarding the genetic resources of apple germplasm in Kazakhstan, the center of origin for apples. In this study, conducted with five apple genotypes [Malus domestica Borkh. and Malus sieversii (Ledeb.) M. Roem] we determined cold hardiness and the effect of cold acclimation on shoot tip recovery following cryopreservation using two techniques. Apple shoot tips were cold acclimated (CA) for 0 to 6 weeks and cryopreserved using PVS2 vitrification and encapsulation dehydration (ED). Cold hardiness was indicated by the temperature at which 50% of the shoot tips were lethally injured (LT50). For non-acclimated shoots, LT50 ranged from -6.7C to -9.3C. These LT50 values resembled the natural cold hardiness of field grown plants and resulted in 10-12% regrowth after cryopreservation. Acclimated plantlets had LT50 values of -12C to -15C after 1 to 3 weeks CA, and after 3 weeks CA, cryopreservation resulted in 65% regrowth. There were no significant differences between the two techniques for regrowth of shoot tips after each cold acclimation period. Overall, 2 to 5 weeks CA produced high regrowth for each of the five cultivars tested. Three weeks of alternating temperature CA can be recommended as a standard protocol for Malus germplasm cryopreservation. These conditions resulted in moderate (60%) to high (80%) recovery for all five genotypes tested with both cryopreservation methods used.

Keywords: acclimation, cold hardiness, germplasm, long-term storage, LT50, Malus domestica, Malus sieversii

 

 

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CryoLetters 30 (1), 55-63 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

CRYOPRESERVATION OF EMBRYOGENIC CULTURES OF 'BREWSTER' LITCHI (Litchi chinensis SONN.) AND ITS EFFECT ON HYPERHYDRIC EMBRYOGENIC CULTURES

Guillermo Padilla, Pamela Moon, Irene Perea and Richard E. Litz*

Tropical Research and Education Center, University of Florida, 18905 S.W. 280 Street, Homestead FL 33031-3314, USA.
*Corresponding author e-mail:
relitz@ufl.edu

Abstract

Cryopreservation of embryogenic cultures induced from leaves of mature phase trees of Litchi chinensis Sonn. was performed following a vitrification method. Vitrification solution (PVS2) was utilized at two temperatures: 0C and 25C. Posttreatment survival percentages and regrowth rates of the cultures were higher when the PVS2 solution was at 0C. All samples cryopreserved with PVS2 at 0C survived; their regrowth rate after eight weeks on semi-solid maintenance medium was the same as non-treated controls. Cryopreservation suppressed somatic embryo development; the number of somatic embryos derived from cryopreserved cultures was less than the number obtained from the controls. Desiccation during the PVS2 treatment had no effect on reversal of hyperhydric embryogenic cultures.

Keywords: cryopreservation, litchi, hyperhydricity, somatic embryo, vitrification.

 

 

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CryoLetters 30 (1), 64-75 (2009)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

CRYOPRESERVATION OF EMBRYOGENIC CALLUS OF Dioscorea bulbifera BY VITRIFICATION

Senrong Hong, Minghua Yin, Xinghua Shao, Aiping Wang and Weihong Xu

Department of Life Sciences, Shangrao Normal College, Shangrao 334001, China. *Correspondence author e-mail: hongsenrong@163.com

Abstract

Cryopreservation of callus of Dioscorea bulbifera by vitrification was optimized. Calli of Dioscorea bulbifera were pretreated in liquid Murashige and Skoog (MS) medium supplemented with 2 mg L-1 kinetin (KT), 0.5 mg L-1 NAA, 0.5 mg L-1 2,4-D and 0.2 M sucrose for 5 d under continuous light (36 μM m-2 s-1) at 251C. The material was then loaded with 60% vitrification solution (PVS2) for 20 min at room temperature and dehydrated with 100% PVS2 for 30 min at 0C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen and conserved for 1-360 d. After rapid thawing in a water-bath at 35C, the calli were washed three times with liquid MS medium supplemented with 2 mg L-1 KT, 0.5 mg L-1 NAA, 0.5 mg L-1 2, 4-D and 1.2 M sucrose and then transferred onto solid MS medium supplemented with KT 2 mg L-1, NAA 0.5 mg L-1, 0.09 M sucrose and 0.75% (w/v) agar. The cultures were kept in the dark for 2 days prior to exposure to the light (12 h light/dark cycle). The TTC test showed that 80-90% of the calli survived this cryoprocedure and there was a 60-70% regeneration of plantlets from the calli. The regenerated material did not exhibit any morphological variations.

Key words: Dioscorea bulbifera, cryopreservation, vitrification, callus

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