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Abstracts: CryoLetters 23 (1), 2002

CryoLetters 23, 5-10 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

FREEZING RATE AFFECTS THE SURVIVAL OF A SHORT-TERM FREEZING STRESS IN Panagrolaimus davidi, AN ANTARCTIC NEMATODE THAT SURVIVES INTRACELLULAR FREEZING

D.A. Wharton1*, G. Goodall1,2 and C.J. Marshall2

Departments of Zoology1 and Biochemistry2, University of Otago, P.O. Box 56, Dunedin, New Zealand.
*Corresponding author, Email: david.wharton@stonebow.otago.ac.nz

Abstract

The ability of the Antarctic nematode Panagrolaimus davidi to survive a short-term freezing stress depended upon the rate of freezing of its surroundings, measured as the duration of the sample exotherm. The freezing rate increased as the sample volume and freezing temperature decreased and resulted in fewer nematodes surviving. This appears to be due to the greater risk of physical damage by ice crystal growth at high freezing rates. Once frozen the nematodes will then survive exposure to lower temperatures. The environment of the nematode is likely to produce the slow rate of freezing of its surroundings that is necessary for its survival.

Keywords: Freeze tolerance, cooling rate, freezing rate, exotherm duration, intracellular freezing

CryoLetters 23,11-20 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

 

 

CryoLetters 23, 11-20 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECTS OF THE CRYOPRESERVATION PROCEDURES ON RECOVERED RICE CELL POPULATIONS

O. Moukadiri1, J.E. O´Connor2 and M.J. Cornejo1*

1Departamento de Biología Vegetal, Universidad de Valencia, Fac. Biología, Dr. Moliner, 50. 46100 Burjassot, Valencia, Spain
Fax: 34-963864372  e-mail: Maria.J.Cornejo@uv.es
2 Departamento de Bioquímica y Biología Molecular, Centro de Citometría Culter y Técnicas Afines, Facultad de Medicina, Universidad de Valencia.

Abstract

Cryogenic storage of plant cells allows the long-term maintenance of valuable genotypes. Cryopreservation of calli and cell suspensions is often performed using cryoprotectants and slow cooling rates. Rice calli (Oryza sativa L.) were cryopreserved by this procedure as well as by direct immersion in liquid nitrogen without cryoprotection. Subsequently, the characteristics of the recovered cells as well as the effects of putative cryoselection were investigated by microscopic observations and flow cytometric analyses. For this purpose, protoplasts were prepared from calli that had been cryopreserved by direct plunging into liquid nitrogen and from their unfrozen controls. Results show that direct immersion in liquid nitrogen of calli pre-treated with abscisic acid is a fast and highly efficient freezing procedure that maintains the main characteristics of the cell populations and appears to increase their metabolic activity.

Keywords: Abscisic acid, cryopreservation, flow cytometry, Oryza sativa L., protoplasts, rice calli.

 

 

CryoLetters 23, 21-26 (2002)
CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

FIELD PERFORMANCE OF SUGARCANE (Saccharum sp.) PLANTS derived from CRYOPRESERVED CALLUSes

M.E. Martínez-Montero1*, E. Ojeda2, A. Espinosa2, M. Sánchez1, R. Castillo1,

M.T. González-Arnao3, F. Engelmann4, 5 and J.C. Lorenzo1

1Centro de Bioplantas, Universidad de Ciego de Avila, CP 69450, Ciego de Avila, Cuba.
2Estación Provincial de Investigaciones de la Caña de Azúcar, Ciego de Avila, Cuba.
3Universidad de La Habana, Fac. Biología, Calle 25 e/ J e I, Vedado, La Habana, Cuba.
4International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
5Institut de recherche pour le développement (IRD), 911 avenue Agropolis, 34032 Montpellier cedex 01, France (current address).
*to whom correspondence should be addressed (email: cubamarcose00@hotmail.com .

Abstract

This study compared the field performance of sugarcane plants originating from three different sources: control, non-cryopreserved embryogenic calluses, cryopreserved embryogenic calluses and macropropagated material of the same commercial hybrid. Several agronomic traits were evaluated on 100 plants per treatment over a 27-month period covering the growth of the stool and of the first ratoon. Significant differences between treatments were observed only during the first six months of field growth of sugarcane stools. Stems produced from in vitro cultured material, irrespective of their cryopreservation status, had a smaller diameter and a shorter height than those produced from macropropagated material. These differences disappeared by12 months of stool field growth.

Keywords: sugarcane; Saccharum sp.; cryopreservation; embryogenic callus; macropropagated material; field performance.

 

 

CryoLetters 23, 27-35(2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECTS OF CRYOPRESERVATION ON DEVELOPMENTAL COMPETENCY, CYTOLOGICAL AND MOLECULAR STABILITY OF CITRUS CALLUS

Yu-Jin Hao1, Chun-Xiang You2 and  Xiu-Xin Deng*

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, P. R. China
1Present address: College of Life Sciences, Peking University, Beijing 100871, P. R. China E-mail: yjcxhao@hotmail.com
2 Present address: Laiyang Agricultural College, Laiyang 265200, Shandong Province, P. R. China
* To whom correspondence should be addressed. E-mail: dxxwwlj@public.wh.hb.cn

Abstract

Cell suspensions of twelve citrus genotypes were successfully cryopreserved by vitrification. All genotypes survived cryopreservation with >90% viability and the surviving cells of some genotypes regenerated somatic embryos better than the controls. Single-cell sibling lines of cultivar Newhall were used for cytological and molecular examination. It was found that the ploidy constitution remained genetically stable and that no DNA sequence variation was detected by randomly amplified polymorphic DNA (RAPD) assay after cryopreservation. In addition, the methylation sensitive amplified polymorphism (MSAP) assay indicated that cryopreservation caused a significant change in DNA methylation status.

Keywords:  vitrification, somatic embryogenesis, cryopreservation,  ploidy, RAPD,  DNA methylation

 

 

CryoLetters 23, 37-46 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ANALYSIS OF PLOIDY AND THE PATTERNS OF AMPLIFIED FRAGMENT LENGTH POLYMORPHISM AND METHYLATION SENSITIVE AMPLIFIED POLYMORPHISM IN STRAWBERRY PLANTS RECOVERED FROM CRYOPRESERVATION

Yu-Jin Hao1,  Chun-Xiang You2 and Xiu-Xin Deng*

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, P.R. China
1Present address: College of Life Sciences, Peking University, Beijing 100871, P.R. China E-mail: yjcxhao@hotmail.com
2 Present address: Laiyang Agricultural College, Shandong 265200, P. R. China
* To whom correspondence should be addressed. E-mail: dxxwwlj@public.wh.hb.cn

Abstract

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.

Keywords: genetic resource, encapsulation-dehydration, regeneration, somaclonal variation, DNA methylation

 

 

CryoLetters 23, 47-54 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION AND XENOTRANSPLANTATION STUDIES OF MICROENCAPSULATED RAT PANCREATIC ISLETS

Bao-Guo Li1*, Tse-Chao Hua1, Hong-De Zhang2, Yu-Fei Wang2, Guo-Xing Wang2

1Institute of Cryobiological Engineering, Shanghai University of Science and Technology, No.516 Jun Gong Road, Shanghai, 200093, China
2Research laboratory of diabetes, Shanghai First Peoples' Hospital, Shanghai, 200080, China

Abstract

Islets of Langerhans were isolated from the Sprague Dawley rat pancreas digested by injected collagenase, and purified by Ficoll density gradient centrifugation. In order to make smaller and more uniform microencapsulated islets, we designed a special high-voltage electrostatic microcapsule generator. The effects of operational parameters of the generator on the size and the uniformity of microcapsules were analyzed, such as the voltage, the plunger speed of suspension delivery to the needle tip, the distance between needle tip and solution surface. The optimal parameter combinations for making microcapsules are: 5kV of voltage, 50mm/h of the plunger speed, and 20mm distance. The high-voltage electric system can produce uniform microcapsules with diameters ranging from 0.3~0.5mm, which are smaller and more uniform than those produced by air-jet system. A comparison of the cryopreservation effects between microencapsulated islets and unencapsulated islets showed that the microcapsules can protect the fragile islets from freezing damage, and increase the retrieval rate from 68.5% to 92.6%. Xenotransplantation of the cryopreserved rat islets resulted in the normalization of the metabolic blood glucose of the diabetic mice for 90 days, whereas the unencapsulated islets were easily fragmented and lost during the freezing process.  They only reversed hyperglycemia for less than 3-5 days.

Keywords: cryopreservation, microencapsulation, rat pancreatic islet, xenotransplantation

 

 

CryoLetters 23, 55-60 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

AMMONIUM NITRATE IN THE CULTURE MEDIUM INFLUENCES REGENERATION POTENTIAL OF CRYOPRESERVED SHOOT TIPS OF Holostemma annulare

S. William Decruse* and S. Seeni

Plant Biotechnology Division, Tropical Botanic Garden & Research Institute, Palode, Thiruvananthapuram-695562, Kerala, India

Abstract

Influence of NH4NO3 in the pre-freeze and post-freeze culture medium and 2 or 30 day preconditioning in the presence of 0.5 M sucrose on regeneration of shoot tips of Holostemma annulare following cryopreservation using an encapsulation-dehydration protocol was studied.  A long preconditioning phase of 30 days significantly reduced tissue water and improved post-freeze recovery of shoot tips. Under the long preconditioning treatment, Murashige & Skoog (MS) medium free of NH4NO3 (MS-3) allowed maximum regeneration (59%) of liquid nitrogen (LN) exposed shoot tips with less frequency of callusing (10.4%) after 45 days of post-freeze culture. Corresponding desiccated control shoot tips showed 85-90% regeneration. A 3.75 mM NH4NO3 concentration (MS-4) favoured 72-89% and 43-47% regeneration after desiccation and LN exposure respectively. The standard MS medium with 20.6 mM NH4NO3 (MS-1) allowed poor regeneration after desiccation (39-53%) as well as LN exposure (8-23%). The study reveals the importance of reducing ammonium nitrate in the culture medium to get maximum recovery of cryopreserved shoot tips of Holostemma annulare.

Keywords: cryopreservation, encapsulation-dehydration, Holostemma annulare, medicinal plant, sucrose, preconditioning.

 

 

CryoLetters 23, 61-68 (2002)
 CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF CULTIVATED AND WILD Arachis SPECIES EMBRYONIC AXES USING DESICCATION AND VITRIFICATION METHODS

R.F. Gagliardi1, G.P. Pacheco1, J.F.M. Valls2 & E. Mansur1*

1Laboratório de Micropropagação e Transformação de Plantas, Universidade do Estado do Rio de Janeiro, Brazil
2EMBRAPA/CENARGEN, Brasília, DF, Brazil
*for correspondence (e-mail: mansur@uerj.br )

Abstract

The effects of two methods of cryopreservation involving chemical vitrification and air desiccation) were studied on isolated embryonic axes of A. hypogaea. Vitrification with PVS2 and desiccation in a laminar flow cabinet resulted in high levels (70-90%) of whole plant recovery after cryopreservation. A desiccation protocol based on 1h exposure of explants to the air flow was successfully applied to six wild species of section Extranervosae, resulting in recovery levels of 70-90% after liquid nitrogen treatment.

Keywords: germplasm preservation, peanut, groundnut, Extranervosae, desiccation, cryopreservation.

 

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