Abstracts: CryoLetters 25 (2), 2004

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

CryoLetters 25, 81-90 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


M. Lambardi1*, A. De Carlo1,2, S. Biricolti2, A.M. Puglia3,
G. Lombardo4, M. Siragusa5 and F. De Pasquale5

Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, via Madonna del Piano, 50019 Sesto Fiorentino (Firenze), Italy.
2 Dipartimento di Ortoflorofrutticoltura, Università degli Studi di Firenze, Polo Scientifico, viale delle Idee, 50019 Sesto Fiorentino (Firenze), Italy.
3 Dipartimento di Biologia e dello Sviluppo, Università degli Studi di Palermo, viale delle Scienze, 90128 Palermo, Italy.
4 Dipartimento di Scienze Botaniche, Università degli Studi di Palermo, via Archirafi 38, 90123 Palermo, Italy.
5 Istituto di Genetica Vegetale, sez. di Palermo, CNR/Consiglio Nazionale delle Ricerche, Corso Calatafimi 414, 90129 Palermo, Italy.
* For correspondence:


A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16% (C. sinensis) or 10% (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50% after drying to 15% moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93% germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.

Keywords: Citrus, cryopreservation, seed, dehydration, nucellar embryos, ISSR



CryoLetters 25, 91-100 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Evolution of DMSO Concentration in Garlic Shoot tips during a Vitrification Procedure

Haeng-Hoon Kim1*, Jung-Bong Kim1, Hyung-Jin Baek1, Eun-Gi Cho1, Young-Am Chae2 and Florent Engelmann3,4

1 National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea (email for H.H. Kim:
2 Department of Agronomy and Research Center for New Biomaterials in Agriculture, Seoul National University, Suwon 441-744, Korea.
3 Cirad-flhor, TA 50/PS4, Boulevard de la Lironde, 34398 Montpellier cedex 5, France (present address).
4 International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


In this paper, the evolution of dimethylsulfoxide (DMSO) concentration and moisture content (MC) of garlic shoot tips was studied during the course of a vitrification protocol using the PVS2 vitrification solution. DMSO concentration of shoot tips increased rapidly, reaching 34.1 mg/g fresh weight after 20 min of PVS2 treatment and remained stable afterwards, while moisture content decreased from 82 to 60 %, reaching 53 % after 60 min. A reverse process was observed during unloading. There was a highly significant negative correlation between shoot tip moisture content and DMSO concentration during the dehydration and unloading treatments. Using unloading solutions with osmolarities between 0.42 and 2.29 Osm led to very different shoot tip MCs, between 63.55 and 81.24 %, while DMSO concentration was between 14.83 and 19.97 mg/g fresh weight. After 24 h on recovery medium, DMSO concentration of shoot tips had decreased to 3.2 mg/g fresh weight.

Keywords: garlic, shoot tips, DMSO, HPLC, PVS2, vitrification.



CryoLetters 25, 101-110 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A COMPARISION OF FOUR METHODS FOR cryopreservation of meristems IN banana (Musa spp.)

Anuradha Agrawal1*, Rony Swennen and Bart Panis

Laboratory of Tropical Crop Improvement, Catholic University of Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium
1Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110 012, India 


To establish an improved protocol for the cryopreservation of banana (Musa spp.), a fast-freeze/fast-thaw method was applied and compared to three existing cryogenic procedures, namely, simple freezing, vitrification of proliferating meristems and vitrification of individual meristems. The average post-thaw shoot regeneration of the fast freeze/fast thaw protocol was 51.8% and was superior to the existing protocols which were 12.5%, 39.2% and 42.8%, respectively. Other factors such as time and ease of operation were considered for each method. It was found that the fast freeze/fast thaw technique was the best amongst all the four methods tested for the cryopreservation of in vitro banana meristems in the genotype Robusta (AAA).

Keywords: banana, benzyladenine, cryopreservation, cryoprotection, meristem, Musa spp., Robusta, sucrose, vitrification



CryoLetters 25, 111-120 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Hong-Hai Xiao1, Tse-Chao Hua1*, Jun Li1, Xue-Lian Gu1, Xin Wang1, Zhi-Jiang Wu2, Lv-Rong Meng2, Qi-Rong Gao2, Jian Chen2 and Zhong-Ping Gong2

1 Institute of Cryomedicine and Food Refrigeration, Shanghai University of Science and Technology, No.516 Jun-Gong Road, 200093, Shanghai, China.
* E-mail:
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science, No.320 Yo-Yang Road, 200031, Shanghai, China


The research on haematopoietic stem cells of human cord blood has become more important recently. People have concentrated on the preservation of cord blood stem cells. At present, cord blood can be preserved at ultra-low temperatures. In this study, we try to preserve cord blood and its constituents by freeze-drying. The experiments on both the mononuclear cell content and the whole blood of human cord blood were carried out respectively. The samples were frozen firstly by different cooling protocols in the presence of PVP, sucrose, and mannitol. Afterwards, they were vacuum-dried at a selected shelf temperature of –30ºC for the main drying stage, and then vacuum-dried at 15ºC for the second drying stage. The entire time of the freeze drying was 52 hours. Samples were stored at room temperature for 2 days prior to evaluation. Subsequently, the dried samples were suspended in an isotonic phosphate-buffered saline solution.
The recovery of the cells were tested by a haemacytometer, and the highest cell numerical count recovery of MNC was 75.0 ± 4.1%(P<0.01), obtained in the protocol of 40% VP + 20% Sucrose + 10% Mannitol. The viability of the nucleated cells measured by PI staining and the ratio of the number of CD34+ to the number of lymphocytes (by the FITC anti-human CD34+ conjugated antibody method) were measured using a flow cytometer (FCM). The protocol of 40% PVP + 20% Sucrose + 10% fetal bovine serum had the highest viability of 98.57±0.68%(P<0.01). The highest ratio of CD34+ to lymphocytes was 1.17%, and the highest recovery of CD34+ was 68.42 ± 39.5% (P<0.05). Comparing the results of the lyophilized MNC subfraction with that of the whole blood, the lyophilization of the isolated MNC was more successful than that of whole blood.

Keywords: freeze-drying, human cord blood, MNC , lyoprotectant, CD34+ cell



CryoLetters 25, 121-128 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


James Wesley-Smith*1, Christina Walters2, Patricia Berjak1 & NW Pammenter1

1 School of Life and Environmental Sciences, University of Natal, Durban, South Africa
2 National Center for Genetic Resources, 1111 S. Mason, Ft. Collins CO 80521, USA


The present study investigated the rate of temperature change within axes of Poncirus trifoliata during cooling and warming by various methods. Cooling rates ranged between 0.17 and 1700C s-1, and warming rates of 1.25 and 600C s-1 were measured when axes were warmed at room temperature or in water at 40ºC, respectively. Partial drying increased the cooling rate within axes in direct contact with the cryogen, but did not affect the cooling or warming rates within axes enclosed in a double layer of lightweight aluminium foil. The procedures described illustrate the orders of magnitude that separate extremes of the range of cooling or warming rates attained using methods commonly employed in cryopreservation studies. Quantifying these rates allows the relationship between cooling rate, water content and survival of hydrated embryonic axes to be explored.

Keywords: embryonic axes, rapid cooling, dehydration, Poncirus trifoliata, warming, recalcitrance, cryopreservation



CryoLetters 25, 129-138 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


James Wesley-Smith*1, Christina Walters2, Patricia Berjak1 and NW Pammenter1

1 School of Life and Environmental Sciences, University of Natal, Durban, South Africa
2 National Center for Genetic Resources, USDA–ARS, 1111 S. Mason, Ft. Collins CO 80521, USA


The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 gH2O g-1 dry mass (g g-1) is believed to increase cytoplasmic viscosity. Cooling to -196ºC was done at rates averaging between 0.17 and 1300ºC s-1, and warming at 600 or 1.35ºC s-1. Survival was assessed after 4 weeks in vitro.  Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70% normal development ranged between 0.17 and about 1300ºC s-1 for axes at a water content of 0.26 g g-1, narrowing with increasing hydration to an apparent optimum at about 686ºC s-1 in axes at 0.8 g g-1. At 1.7 g g-1, axes cooled at 0.17ºC s-1 yielded nearly 40% normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.

Keywords: embryonic axes, rapid cooling, cytoplasmic viscosity, Poncirus trifoliata, warming, recalcitrant seed, water content, cryopreservation



CryoLetters 25, 139-146 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


W.K. Berger

Physiol. Institut, Bau 59, Unikliniken 66421 Homburg, Deutschland.


Ice-cell interactions especially the question whether and how ice crystals invade tissues and organs during freezing were examined in small transparent organs (salivary glands) in which many structural details remained visible despite of freeze-induced cell darkening. In most glands no invasion of ice into the lumen was observed since ice dendrites stopped growing after touching the gland.  Here I report that in rare cases a so far unknown type of ice crystals developed which aggressively pushed against cell membranes before invading the gland via paracellular pathways (septate junctions). Aggressive ice crystals were also observed within a salivary gland cell which deformed and finally invaded the nucleus. In cell strands it was observed that intracellular freezing is indeed a two-step event in which ice developed in cytoplasm several seconds before invading the nucleus.

Keywords: ice crystal growth, salivary gland, septate junctions, paracellular pathways, Chironymus thummi



CryoLetters 25, 147-154 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


A. Akourki1, L. Gil1*, A. Echegaray3, E. Espinosa1, A. Josa1, I. de Blas1, N. Gonzalez1, M. Gallegos de la Hoya2 and L.C. Meque1

1 Facultad Veterinaria, Depto. de Patología Animal (Reproducción), Universidad de Zaragoza, c/ Miguel Servet 177, 50013 Zaragoza, Spain.
2 Facultad de Medicina Veterinaria y Zootecnia, Universidad de Juárez del Estado de Durango, 34000 Durango, Mexico.
3 Empresa Magapor SL., Ejea de los Caballeros, 50600 Zaragoza, Spain


This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser's extender, to which 0.7 % Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p<0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37ºC after thawing, Equex-STM also showed a beneficial effect on sperm quality.

Keywords: Assaf sheep, semen, Equex-STM, extender, cryopreservation

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