Abstracts: CryoLetters 22 (6), 2001

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

CryoLetters 22, 341-352 (2001)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Matsuo Uemura

Division of Biosystem and Bioresource Technology, Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan

Immediately after starting my graduate study at the Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan, in 1979, my supervisors began mentioning to me the name of a renowned cryobiologist who had published many important papers over a number of years..  That name was Peter L. Steponkus. I met Pete for the first time in 1981 and following from that, I had fortunately had a long collaboration with Pete until his death, including my stay of more than 10 years in his laboratory at Cornell University.  I learned from Pete a lot about the way to carry out scientific research. I would like to share my thoughts about Pete’s contribution to the areas of plant and membrane cryobiology, for which he will long be remembered.



CryoLetters 22, 353-366 (2001)
 Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK


M.S. Berwane and J.C. Bischoff

Mechanical Engineering Department, Urologic Surgery and Biomedical Engineering Department, University of Minnesota Minneapolis, Minnesota 55455


There is mounting evidence that the endothelium may play an important role in traditional cryosurgical treatments by acting to locally foster thrombi in the microvasculature of various tissues after freezing.  In addition, new catheter based cryosurgical probes are being designed for cardiovascular applications where endothelial and smooth muscle cell freezing is involved but not well understood.  Therefore, this study was designed to investigate, at the cellular level in human microvascular endothelial cells (hMEC), the various biophysical changes that occur during freezing which can affect post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics at 5, 10 and 25 °C/min were found to be governed by a membrane permeability Lpg and activation energy ELp of 0.05 (m/min.atm) and 14.8 (kcal/mole) respectively [R2=0.94].  These parameters were then tested for predictive ability against the experimentally measured behavior at 15C/min with a good agreement [R2=0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% ~ -18C) and at cooling rates greater than or equal to 25C/min.  At cooling rates above 50C/min, two types of IIF, cell darkening and twitching, were both observed and quantified and were assumed to be governed by Surface Catalyzed Nucleation (SCN).  IIF parameters, o and o, which fit data from 50, 100 and 130 °C/min were found to be 6.8 x 10-8 (m2.s)-1 and 8.3 x 10-9 (K5) [R2=0.94] respectively.   Preliminary results show that viability drops precipitously between –20 and –30 °C, however, further studies are warranted to address the role of cooling rate, end-temperature, hold time and thawing rate to establish the freeze sensitivity of this cell.

Keywords: Endothelial cells, freezing, biophysics, cryomicroscopy, water transport, intracellular ice formation



CryoLetters 22, 375-380 (2001)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of apple dormant buds and shoot tips

Yongjie Wu1*, Yanhua Zhao1, Florent  Engelmann2, 3, Mingde Zhou4, Deming Zhang1 and Shuangying Chen1

1Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Hebei 066600, Changli Town, China. (email:
2IPGRI, Via dei Tre Denari, 472/a, 00057 Maccarese (Fiumicino) Rome, Italy.
3IRD, BP 5045, 34032 Montpellier Cedex 01, France (present address).
4IPGRI, East Asia Office, c/o CAAS, 30 Bai Shi Qiao Road, Beijing100081, China.


Shoot tips excised from apple (Malus domestica Borkh. cv. Golden Delicious) dormant buds immediately before freezing were successfully cryopreserved using three different methods (vitrification, encapsulation-dehydration  and two-step freezing). Two-step freezing produced the highest in vitro growth recovery (83%) of cryopreserved shoot tips, followed by vitrification (60%) and encapsulation-dehydration (33%). By contrast, when dormant buds were cryopreserved using the two-step freezing technique, shoot tips excised from buds after rewarming and cultured in vitro displayed only 16% recovery.

Keywords: Malus domestica; dormant buds; shoot tips; cryopreservation.



CryoLetters 22, 367-374 (2001)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


M. Al-Forkan1, P. Anthony, J.B. Power, M.R. Davey and K.C. Lowe2*

Plant Science Division, School of Biosciences, 2School of Life & Environmental Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
Present Address: Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.
*To whom correspondence should be addressed (email


This study shows that adding haemoglobin solution (Erythrogen™) to post-thaw medium of Indica rice (Oryza sativa L.) cells enhances survival following cryopreservation. Haemoglobin (1:50 - 1:200 v:v) had a beneficial effect on post-thaw viability and subsequent cell growth.  A key finding was that the successful recovery from cryopreservation of cell suspensions of the Indica rice cvs. BR26 and Pajam, and their re-establishment in AA2 medium, reflected a requirement for such supplementation of the post-thaw recovery medium with Erythrogen™.

Keywords: Erythrogen™, haemoglobin, cryopreservation, cell suspensions, rice.



CryoLetters 22, 381-389 (2001)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Joyce C. Pennycooke1 and Leigh E. Towill2*

1Department of Horticulture and Landscape Arch., Colorado State University, Fort Collins, CO 80523, USA.
USDA-ARS, National Seed Storage Laboratory, 1111 S. Mason St., Fort Collins, CO 80521 USA.
Email:   * To whom correspondence should be addressed


In vitro grown sweet potato (Ipomoea batatas {L.} Lam.) shoot tips were successfully cryopreserved by both solution based and encapsulation-dehydration vitrification methods. Improved recovery medium enhanced recovery for both vitrification procedures. The effects of sucrose preculture, cryoprotectant preculture and post-warm recovery media on regrowth following LN exposure were investigated. Sucrose preculture was critical for the survival of sweet potato shoot tips cooled to ca. –200ºC. Cryoprotectant preculture with 2 M glycerol + 0.4 M sucrose before dehydration with PVS2 gave the highest recovery following LN exposure.  The viability of cooled samples following culture on ammonium-free MS medium for 5 days was increased three-fold over those cultured on MS medium. The improvement in recovery by altering post-warming conditions suggests that cryoinjury is not always lethal and can be ameliorated by suitable culture conditions.

Key words: Sweet potato, cryopreservation, vitrification, encapsulation-dehydration, recovery medium



CryoLetters 22, 391-396 (2001)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF Citrus madurensis zygotic EmbryoNIC AXES BY VITRIFICATION: Importance of Pregrowth and preculture conditions

Eun Gi Cho1, 3*, Yue Luan Hor 2, Haeng Hoon Kim3 , V. Ramanatha Rao1 and Florent Engelmann4, 5

1International Plant Genetic Resources Institute (IPGRI), Regional Office for Asia, Pacific and Oceania, P.O. Box 236, UPM Post Office, 43400 Serdang, Selangor Darul Ehsan, Malaysia.
2Department of Crop Science, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia.
3National Institute of Agriculture and Science Technology, RDA, Suwon 441-707, Republic of Korea. (current address for E.G.C.;email:
4International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
5Institut de recherche pour le développement (IRD), BP 5045, 34032 Montpellier Cedex 01, France. (current address).


The role of pregrowth and preculture treatments in terms of both medium composition and exposure duration on survival of embryonic axes of Citrus madurensis after cryopreservation using the vitrification procedure was investigated. The optimal pregrowth treatment for excised embryonic axes was a 3-day treatment with 0.1M sucrose. Preculture was also essential in increasing survival after cryopreservation. Among the various media and treatment durations evaluated, a 24h-preculture of embryonic axes on medium with 0.3M sucrose and 0.5M glycerol was found to be optimal. Using these pregrowth and preculture conditions followed by treatment at 25°C for 20 min each with a loading solution (0.4M sucrose + 2.0M glycerol) and then the PVS2 vitrification solution, direct immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2M sucrose solution for 20 min and transfer of embryonic axes on recovery medium, 82.5% survival and regrowth without intermediary callus formation were obtained with C. madurensis embryonic axes.

Keywords: Citrus madurensis; embryonic axes; cryopreservation; vitrification; pregrowth; preconditioning.


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