Abstracts: CryoLetters 25 (1), 2004

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

CryoLetters 25, 3-22 (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK




Reproductive Science and Integrated Conservation

Edited by William V. Holt, Amanda R. Pickard, John C. Rodger and David E. Wildt
Cambridge University Press; ISBN: 0-521-01110-8. February 2003



Keith Harding

Conservation and Environmental Chemistry Centre, School of Contemporary Sciences, Kydd Building, University of Abertay Dundee, Bell Street, Dundee DD1 1HG Scotland UK.


Cryopreservation techniques exist for some 100-plant species. Cryopreservation for the long-term conservation of in vitro germplasm results in the exposure of tissues to physical, chemical and physiological stresses causing cryoinjury. Although, the effects of cryoinjury upon the genome are often unknown, any accumulative DNA polymorphisms may not be induced by cryopreservation per se but are the result of the whole culture-cryoprotection-regeneration process. It is desirable to assess the genetic integrity of plants surviving cryogenic storage to determine if they are ‘true to type’ after cryopreservation.  This can be done at the phenotypic, histological, cytological, biochemical and molecular levels. The relevance of these approaches to stability investigations is discussed with their limitations. This review provides a definition for ‘Cryobionomics’ - a novel term describing the re-modelled concept of genetic stability and the re-introduction of cryopreserved plants into the environment.

Key words: conservation, cryobionomics, cryopreservation, genetic stability, plants



CryoLetters 25, 23-32  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Mushroom cryopreservation and its effect on survival, yield and genetic stability

S. K. Singh*, R. C. Upadhyay, Shwet Kamal and Mugdha Tiwari

National Research Centre For Mushroom (ICAR), Chambaghat, Solan (HP)-173213 India (*


Mycelial stock cultures of Agaricus bisporus, A. bitorquis, Pleurotus flabellatus, P. sajor-caju, P. ostreatus, P. sapidus, Auricularia polytricha, Lentinula edodes, Morchella esculenta and Volvariella volvacea were maintained by frequent subculturing at an interval of two months and separately as wheat grain spawn in liquid nitrogen with 15% glycerol. Preservation of mushroom stock cultures as wheat grain spawn under liquid nitrogen proved to be the better method of maintenance. The percent recoveries of stored samples were unchanged from the first recovery after six months to the last recovery after 42 months in nine out of 11 stock cultures preserved under liquid nitrogen. However, a marginal decline in survival of 10 % was recorded in Auricularia polytricha and Volvariella volvacea. Yields before preservation of mushroom stock cultures and after 30 months of preservation exhibited static biological efficiency and fruitbody weight. The comparison of Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacers (ITS) PCR amplified products did not exhibit DNA fragment variation in banding patterns at the intraspecific level during preservation of stock cultures by either method. The modified cryopreservation protocol and experimental demonstration of genetic stability of stock cultures reported here validate the use of mushroom cryopreservation techniques and supports studies on genetic stability of preserved biological materials.

Key words: mushroom, cryopreservation, RAPD, ITS, genetic stability



CryoLetters 25, 33-42  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of zygotic embryo axes and somatic embryos of european chestnut

Elena Corredoira, M. Carmen San-José, Antonio Ballester and Ana M. Vieitez*

Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain. *E-mail:


This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93% and 100% of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24% (on a fresh weight basis), and some 63% subsequently developed as whole plants. Desiccation to moisture contents less than 19% produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6-8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25% before storage in LN, the embryogenesis resumption level after thawing was 33%. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68%.

Keywords: Castanea sativa, cryogenic storage, dehydration, liquid nitrogen, somatic embryogenesis, vitrification



CryoLetters 25, 43-50  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Zi-Cheng Wang1 and Xiu-Xin Deng*

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, P. R. China.
1Present address: College of Life Science, Henan University, Kaifeng, 475001ôP. R. China
*To whom correspondence should be addressed. E-mail:


A commercial citrus scion cultivar, ‘439’ tangor [C. suavissima Hort. et Tanaka×C. sinensis (L.) Osbesk cv.Gailiangcheng] was used to investigate whether GSH (reduced form of glutathione) could improve survival of a vitrification procedure. The optimal pre-growth treatment was a 3-day pre-culture on basal pre-culture medium (BPM: MT basal medium containing 0.5 mol/L glycerol and 5% sucrose at pH 5.8). GSH of 40 mg/L in the pre-culture medium improved shoot tip survival after cryopreservation. GSH in the recovery medium also improved survival, with 10 mg/L giving the best result. GSH of 40 mg/L in the loading and vitrification solutions also improved survival. The optimal cryopreservation protocol was successfully applied to 12 other citrus cultivars. This is the first report on the successful cryopreservation of shoot-tips from commercial citrus scion cultivars.

Key words: citrus, cryopreservation, vitrification, GSH



CryoLetters 25, 51-58  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Rommanee Charoensub1* Dai Hirai2 and Akira Sakai3

1Scientific Equipment Center, Kasetsart University Research and Development Institute, Bangkok 10900, Thailand. (E-mail:
2Hokkaido Central Agricultural Experiment Station, Naganuma, 069-1395, Japan.
3Hokkaido University (retired), Asabucho 1-5-23, Kitaku, Sapporo 001-0045, Japan.
* to whom correspondence should be addressed


Shoot tips of cassava (Manihot esculenta Crantz) in vitro plantlets were successfully cryopreserved using the encapsulation-vitrification technique. Nodal cuttings of 5 mm length with one leaf were cultured on modified MS medium in Petri dishes (90 mm x 20 mm) for about 28 days. Excised shoot tips were precultured on sucrose enriched (0.3 M) medium for 16 h, encapsulated and osmoprotected with a mixture of 2 M glycerol and 0.6 M sucrose for 90 min at 25 oC before dehydration with PVS2 at 0 oC for 4 h, then plunged in liquid nitrogen. Successfully vitrified shoot tips resumed growth within 3 days, without intermediary callus formation, and developed shoots. Shoot tips sampled from 21 day-old plantlets produced the highest survival of 80%. The percentage survival of vitrified shoot tips differed from 38 to 80% depending on the day of excision. The protocol was successfully applied to four cultivars of cassava with about 80% average percentage of survival. 

Keywords: cassava, cryopreservation, encapsulation-vitrification



CryoLetters 25, 59-70  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of garlic shoot tips by vitrification: Effects of dehydration, Rewarming, unloading and regrowth conditions

Haeng-Hoon Kim1, Eun-Gi Cho1*, Hyung-Jin Baek1, Chang-Yung Kim1,
E.R. Joachim Keller2 and Florent Engelmann3, 4

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea (email for E.G. Cho:
2Institute of Plant Genetics and Crop Plant Research, IPK, Corrensstrasse 3, 06466 Gatersleben, Germany.
3Cirad-Flhor, TA 50/PS4, Boulevard de la Lironde, 34398 Montpellier cedex 5, France. (present address).
4International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92% regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 °C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/l zeatin and 0.3 mg/l gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95%.

Keywords: cryopreservation; garlic; vitrification; dehydration; unloading; warming; regrowth.



CryoLetters 25, 71-80  (2004)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Leigh E. Towill *1 and Mark Widrlechner2

1 USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO 80521,
2 USDA-ARS North Central Regional Plant Introduction Station, Iowa State University, Ames, IA 50011


Twigs of Salix species are candidates for cryopreservation procedures because they become tolerant of freezing temperatures during mid-winter.  We examined several variables in developing a two-step cryopreservation procedure for sections from these twigs. Samples of Salix triandra cooled to -30ºC or -35ºC and then transferred to the vapor phase over liquid nitrogen gave the greatest percent shoot formation.  Cooling rate to 35ºC had a major influence on shoot formation.  Samples cooled at rates greater than 10ºC/hr showed no shoot formation. The highest percent of shoot formation was achieved by cooling at 0.21ºC/hr. Cooling rate from -35ºC to liquid nitrogen did not influence shoot formation. Warming procedures affected shoot formation. Transferring samples from -160ºC to either a +2ºC cold room or to -3ºC methanol gave similar levels of shoot formation. No shoot formation occurred either with warming in +40ºC water or very slowly in a Styrofoam box.  Eight of eleven Salix taxa tested using the established protocol had significant levels of shoot formation after cryogenic treatment.

Keywords: Salix, willow, cooling rate, warming rate, regrowth, shoot formation, root formation.

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