Abstracts: CryoLetters 27 (3), 2006

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

CryoLetters 27 (3), 133-142 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Rajnesh Sant1*, Mary Taylor1 and Anand Tyagi2

1 Regional Germplasm Center, Secretariat of the Pacific Community, Private Mail Bag, Suva, Fiji.
2 Department of Biology, The University of the South Pacific, P.O. Box 1168, Suva, Fiji.
* Author to whom correspondence should be addressed. Email:


In vitro shoot-tips of three cultivars of tropical taro (Colocasia esculenta var. esculenta (L.) Schott) were successfully cryopreserved by vitrification. Different conditioning treatments were required for each of the cultivars, while the vitrification protocol was constant for all. For the cultivars E399 and CPUK, shoot-tips from three-month-old in vitro plants grown on solidified MS were preconditioned on MS with 0.3 M sucrose in the dark for 16 h at 25°C. For the cultivar TNS, donor plants were preconditioned on solid MS with 90 g L-1 sucrose for seven weeks before cryopreservation.  For vitrification, the shoot-tips were loaded with a solution of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C, dehydrated with PVS2 for 12 min at 25°C and plunged in liquid nitrogen. Vials were warmed by rapid shaking in a water bath at 40°C for 1 min 30.  Shoot-tips were rehydrated in liquid MS with 1.2 M sucrose for 15 min at 25°C then plated on recovery medium. Shoot-tips resumed growth within a week and developed into plantlets six to eight weeks later without any callus formation. The best mean recoveries for the three cultivars were 21, 29 and 30% for E399, CPUK and TNS, respectively. This protocol was evaluated with five other taro cultivars with no success. However, this study has shown that vitrification has potential for cryopreserving tropical taro.

Keywords: shoot-tips, cryopreservation, vitrification, tropical taro [Colocasia esculenta var. esculenta (L.) Schott], preculture, preconditioning



CryoLetters 27 (3), 143-154 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of Garlic Bulbil primordiA by the droplet-vitrification procedure

Haeng-Hoon Kim1*, Joung-Kwan Lee2, Ju-Won Yoon1, Jae-Jun Ji2, Sang-Sik Nam3, Hae-Sung Hwang1, Eun-Gi Cho1 and Florent Engelmann4,5

1*National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea. (Correspondence:
2Danyang Garlic Experiment Station, Danyang 441-744, Korea.
3Mokpo Experiment Station, RDA, Muan, 534-833, Korea.
4Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address).
5International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1-95.0 % and 82.7-85.0 %, respectively) were achieved after preculture for 2-4 days at 10°C on solid medium with 0.1-0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 µl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40°C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4-95.4 %.

Keywords: Allium sativum L., droplet-vitrification, bulbil primordium, cooling, warming.



CryoLetters 27 (3), 155-168 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Maria Teresa Gonzalez-Arnao1* and Florent Engelmann2, 3

1Universidad Veracruzana, Facultad de Ciencias Químicas. Prol. OTE. 6, No. 1009, CP 94340, Apartado Postal 215, Orizaba, Veracruz, México.
* To whom correspondence should be addressed
2Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address). 
3International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


Encapsulation-dehydration is a cryopreservation technique based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allowsthe subsequent application of very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents which would be higly damaging or lethal to non-encapsulated samples. An encapsulation-dehydration protocol comprises the following steps: pretreatment, encapsulation, preculture, desiccation, freezing and storage, thawing and regrowth. Encapsulation-dehydration has been applied to around 40 different plant species. The optimization of the successive steps of the encapsulation-dehydration protocol is illustrated for sugarcane apices.

Keywords: encapsulation; dehydration; cryopreservation; germplasm conservation; vegetatively propagated species.



CryoLetters 27 (3), 169-178 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

NEW DETERMINANTS for tolerance OF COFFEE (Coffea arabica L.) seeds to liquid nitrogen exposure

S. Dussert1* and F. Engelmann2

1 IRD, UR 141, 911 Av. d'Agropolis, BP 64501, F-34394 Montpellier, France
2 Pôle Biodiversité Antilles, Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies


The present work establishes for the first time that tolerance of coffee seeds to liquid nitrogen (LN) exposure depends on the initial quality of the seedlot and on the rewarming regime employed. Seedlot quality was estimated by the parameters of a quantal response model of desiccation sensitivity developed previously. The percentage of seedlings recovered from cryopreserved seeds was very well correlated with the relative humidity (RH) at which 90% of the initial viability was retained, RH90, as estimated by the model. Whatever the cooling regime employed, rewarming the seeds slowly by exposing them to ambient air was highly detrimental. Slow rewarming-induced viability loss was not due to imbibitional damage since seeds pre-heated at 37°C after slow rewarming to 0°C exhibited a survival percentage lower than seeds thawed rapidly to 0°C before sowing. The optimal hydration status for coffee seed cryopreservation was also re-examined. Drying seeds in 81%RH provided survival percentages considerably higher than those obtained using the drying RH always employed until now, i.e. 78%. A new procedure for slowly precooling the seeds prior to immersion in LN was also established. It consisted of placing the vials containing the seeds in a dry ice-bath for 25 min. Using this procedure in combination with seed drying in 81% RH and rapid rewarming in a 37°C water-bath for 30 min ensured the highest survival percentages ever obtained with coffee seeds, i.e. 89%, a value which was not significantly different from the initial viability percentage.



CryoLetters 27 (3), 179-184 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


K. Faszer1, D. Draper2, J.E. Green 2, G.J. Morris 1* and B.W.W. Grout2

1Asymptote Ltd., St John’s Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK
2Postgraduate School, Writtle College, Chelmsford, CM1 3RR, UK
*Corresponding author, e-mail:


A Stirling Cycle freezer has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Horse semen samples were cooled in 0.25 ml straws and 15 ml bags in the Stirling Cycle freezer under laboratory conditions and as a portable device, powered from a car battery. For comparison, straws were frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, motility and viability of samples frozen in the Stirling Cycle freezer were not significantly different when compared to samples frozen in the liquid nitrogen freezer. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen.  The freezer has potential for use in veterinary and genetic conservation applications.

Keywords: Cryopreservation, liquid nitrogen-free, spermatozoa, Stirling Cycle freezer, portability, bag freezing



CryoLetters 27 (3), 185-199 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

The parameter conversion from the Kedem-Katchalsky model into the two-parameter model

Songpol Chuenkhum and Zhanfeng Cui*

*Department of Engineering Science, Oxford University, Parks Road, Oxford OX1 3PJ, UK. E-mail:


Cryopreservation is an important process for preserving cells and tissues. The process itself can, however, cause damage to cells and tissues.  During addition and removal of a cryoprotective agent (CPA), cells are subjected to imbalanced osmotic pressures between the intracellular and extracellular solutions. Cells can be injured if these shrinkage and swelling events are beyond their tolerable limits. The characteristics of the cell volume changes during these processes depend on the types of CPA, the methods of adding and removing of the CPA and the permeability of cells to CPA and water.  The typical models of this transportation are the two-parameter (2-p) model and the Kedem-Katchalsky (K-K) model. The K-K model is more general than the 2-p model.  However, there is evidence that in many cases water and CPAs do not permeate through common pathways, hence the use of the reflection coefficient in the K-K model may be unnecessary and in some cases it may create conceptual errors. Therefore, the 2-p model is more suitable for use as a transport model than the K-K model.  The aim of this study is to use the values of the K-K model parameters from data in the literature to calculate the corresponding parameters for the 2-p model. The results from these simulations demonstrate that the cell volume changes during CPA addition and removal from the K-K model can be closely estimated by the 2-p model. 

Keywords: Kedem-Katchalsky model, two-parameter model, hydraulic conductivity, CPA permeability, reflection coefficient.

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