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Abstracts: CryoLetters 28 (4), 2007

CryoLetters 28 (4), 225-240 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF PICEA SITCHENSIS (SITKA SPRUCE) EMBRYOGENIC SUSPENSOR MASSES

Samantha Galea,b Allan Johnb and Erica E Bensona,c*

a Plant Conservation Group, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG, UK.
bForest Research, Northern Research Station, Roslin, Midlothian, UK EK125 957.
c*Current  Address/Author for Correspondence: Research Scientists, Conservation, Environmental Science & Biotechnology, Damar, Drum Road, Cuparmuir, Fife, KY15 5RJ, UK.
 
e.benson-damar@tiscali.co.uk

Abstract

A cryopreservation method comprising sorbitol pre-growth treatment, DMSO cryoprotection and two-step controlled rate cooling has been optimized for differentiated embryogenic suspensor masses (ESM) of Picea sitchensis. The protocol was applied to clonal cultures from five different half-sibling families each represented by five different genotypes and their responses to cryopreservation assessed over 3 years. Nineteen of the 25 clonal lines tested survived LN and were capable of regrowth and producing stage 2-4 somatic embryos. Following the second subculture cycle of ESM after they had been retrieved from cryogenic storage, post-cryopreservation regrowth was comparable with that of controls. A General Linear Model, multifactorial Main Effects Plot revealed no significant differences between genotypes (P>0.05) in response to cryopreservation, whereas significant differences between families (P<0.05) were detected.

Keywords: clonal forestry, somatic embryos, cryopreservation, genebanks

 

 

CryoLetters 28 (4), 241-252 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF IN VITRO GROWN SHOOTS OF GINGER (ZINGIBER OFFICINALE ROSC.)

G. Yamuna, V. Sumathi, S.P. Geetha, K. Praveen, N. Swapna and K. Nirmal Babu*

Division of Crop Improvement & Biotechnology, Indian Institute of Spices Research, Calicut- 673 012, Kerala, India.
* Corresponding author (
nirmalbabu30@hotmail.com)

Abstract

  An efficient cryopreservation technique for in vitro grown shoots of ginger (Zingiber officinale Rosc) was developed based on encapsulation dehydration, encapsulation vitrification and vitrification procedures. Pregrowth and serial preculture were needed to obtain the best regrowth for all techniques. The vitrification procedure resulted in higher regrowth (80%) when compared to encapsulation vitrification (66%) and encapsulation dehydration (41%). In the vitrification procedure shoots were: precultured in liquid Murashige-Skoog medium containing 0.3 M sucrose for 3 days;  cryoprotected with a mixture of 5% DMSO and 5% glycerol for 20 min at room temperature; osmoprotected with a mixture of 2 M glycerol and 0.4 M sucrose for 20 min at 25ºC; before being dehydrated with a highly concentrated vitrification solution (PVS2) for 40 min at 25ºC. The dehydrated shoots were transferred to 2 ml cryotubes, suspended in 1 ml PVS2 and plunged directly into liquid nitrogen. In all the three cryopreservation procedures tested, shoots grew from cryopreserved shoot tips without intermediary callus formation. The genetic stability of cryopreserved ginger shoot buds were confirmed  using ISSR and RAPD profiling.

Keywords: conservation, cryopreservation, dehydration, encapsulation, genetic stability, germplasm, ginger, ISSR, RAPD, vitrification.

 

 

CryoLetters 28 (4), 253-260 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

MICRO-COMPUTED TOMOGRAPHY OBSERVATION OF SUBLIMATION INTERFACE AND IMAGE ANALYSIS ON SUBLIMATION PROCESS DURING FREEZE-DRYING

Xin Xiao, Le-Ren Tao* and Tse-Chao Hua

Institute of Cryomedicine and Food Refrigeration, University of Shanghai for Science and Technology, No.516 Jun-Gong Road, 200093, Shanghai, China
Tel: 86-21-55270734 Fax: 86-21-55272403
E-mail:
cryo307@usst.edu.cn

Abstract

The freeze-drying process is complicated with complex heat and mass transfer during sublimation. The sublimation interface of freeze-drying has become more attractive and meaningful recently. In this study, apple slices undergoing sublimation were scanned by a Micro-CT scanner. The cross-sectional images were reconstructed with those scanning images respectively. The technique of grey value analysis was used to recognize the procedure. The results showed that, from direct scanning images and 2-D reconstructed images, a 3-D moving mode of sublimation interface which contracted to the centre of the sample could be seen, sublimation process proceeded from edge to center gradually. The grey value of ice crystals was determined to be 154 through Gauss calculation. By comparing frozen sample with freeze-dried one, the ice crystals regions in the beginning became the porous regions after drying, grey values increased correspondingly. Samples shrunk slightly after drying for 3~7 hours, which could be distinguished by the change in grey values.

Keywords: freeze-drying, sublimation process, Micro-CT, phase transformation

 

 

CryoLetters 28 (4), 261-270 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

PHYSICAL STATES OF AQUEOUS SOLUTIONS OF OXYETHYLATED GLYCEROL WITH POLYMERIZATION DEGREE OF N = 30 AT TEMPERATURES LOWER THAN 283 K

Elena N. Zhivotova1*, Alexandra V. Zinchenko2, Larisa G. Kuleshova2, Valentina V. Chekanova2 and Antonina M. Kompaniets2

1National University of Pharmacy, 61002, 53, Pushkinskaya str., Kharkov, Ukraine.
2Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, 61015, 23, Pereyaslavskaya str., Kharkov, Ukraine.
*Corresponding author: E-mail:
zhivotova@inbox.ru

Abstract

A binary system water–oxyethylated glycerol with polymerization degree of n=30 (OEGn=30) is studied over the concentration range of 0-100% (w/w) and the temperature range of 123-283 K by differential scanning calorimetry (DSC). A phase diagram of this system is constructed at the average cooling rate of 200 К min–1 and the warming rate of 0.5 К min–1. Ice crystallization occurs and 70% (w/w) unfrozen residues remain during cooling in the OEGn=30 concentration range of 0-45% (w/w). The system hardens in the amorphous state during cooling and ice crystallization occurs during warming in the range of 46-62% (w/w). Crystallization is observed neither during the cooling nor the subsequent warming in the range of 63-74% (w/w). OEGn=30 crystallization occurs in the range of 75-100% (w/w). Each OEGn=30 molecule can strongly bind to 48 water molecules. In parallel with DSC study, the water–OEGn=30 system is investigated by cryomicroscopy in the temperature range of 173-283К. Cryomicrographs of the solidified solutions representing each of the four concentration bands mentioned above are given.

Keywords: oxyethylated glycerol, DSC, cryomicroscopy, low temperatures, phase transitions, glass transition

 

 

CryoLetters 28 (4), 271-280 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

SURVIVAL OF BOVINE FIBROBLASTS AND CUMULUS CELLS AFTER VITRIFICATION

Barbara Gajda*, Lucyna Kątska-Książkiewicz, Bożenna Ryńska,
Michał Bochenek and Zdzisław Smorąg

Department of Animal Reproduction Biotechnology, National Research Institute of Animal Production, 32-083 Balice/Kraków, Poland.
E-mail:
bgajda@izoo.krakow.pl

Abstract

The aim of the experiment was to investigate the effect of vitrification on viability and the cell cycle of bovine cumulus cells and fibroblasts after culture with or without serum starvation. In all vitrified-thawed bovine somatic cells, the number of samples that reached the confluence stage was high (50 to 100%). The viability of vitrified somatic cells depended on the concentration of the cells. The viability was higher for cells vitrified at the concentration of 10 x 106/ml than for cells vitrified at a concentration of 1 x 106/ml (P<0.05; for cumulus cell, and fibroblast). Time of cell starving has had no impact on their susceptibility to vitrification in case of vitrified cumulus cells. Starving time caused shifts in proportions of subsequent cell cycle phases of vitrified fibroblasts and cumulus cells. In conclusion, the bovine cumulus and fibroblast cells can be cryopreserved successfully by vitrification procedure.

Keywords: cryopreservation, vitrification, cattle, cumulus cells, skin fibroblast, serum starvation, cell cycle

 

 

CryoLetters 28 (4), 281-290 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF SOMATIC EMBRYOS OF PARADISE TREE (MELIA AZEDARACH L.)

Adriana Scocchi1, Silvia Vila1, Luis Mroginski1* and Florent Engelmann2, 3

1 Facultad de Ciencias Agrarias (UNNE), IBONE (Instituto de Botánica del Nordeste), CC 209, Corrientes (3400), Argentina.
(E-mail:
luis@agr.unne.edu.ar).
2 Institut de recherche pour le développement (IRD), UMR DIA-PC, 911 avenue Agropolis, BP 64501, F-34394 Montpellier cedex 5, France.
3 Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.

Abstract

In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25°C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M / 0.75 M / 1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1°C/min from 20°C to -30°C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.

Keywords: immature fruits, zygotic embryos, encapsulation-dehydration, pregrowth-dehydration, sucrose, desiccation, two-step freezing.

 

 

CryoLetters 28 (4), 291-302 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ACQUISITION AND LOSS OF CRYOTOLERANCE IN LIVISTONA CHINENSIS EMBRYOS DURING SEED DEVELOPMENT

Bin Wen1,2* and Songquan Song1

1Xishuangbanna Tropical Botanical Garden, the Chinese Academy of Sciences, Mengla, Yunnan, 666303, China.
 2Graduate School of the Chinese Academy of Sciences, Beijing, 100039, China
*Author for correspondence (E-mail:
wenb@xtbg.org.cn)

Abstract

Changes in desiccation tolerance and cryotolerance of Chinese fan palm (Livistona chinensis [Jacq.] R. Br.) embryos were studied during seed development from 15 to 45 weeks after flowering (WAF). Acquisition and then progressive loss in both desiccation tolerance and cryotolerance was observed within this period. Survival (apparent elongation of embryos) and emergence (formation of root and/or shoot) of embryos following dehydration increased progressively with development of seeds until 33 WAF, and then decreased up to 45 WAF. Similar changes occurred in the minimum moisture content at which 90% of embryos survived or emerged. Cryotolerance of embryos was nil at the early stages of seed development, until 21 WAF. Embryos acquired slight cryotolerance at 23 WAF and cryotolerance increased gradually from 27 to 36 WAF, then decreased by 45 WAF. Survival and emergence of post-thaw embryos were closely related to their moisture contents prior to freezing. However, this correlation between cryopreservation and moisture content was notably influenced by the embryos' developmental stage. Embryos at stages with greater cryotolerance gave higher post-thaw survival and emergence at a given moisture content, and the moisture content range allowing embryos to avoid cryo-damage was widened at both the lower and upper limits. Greater than 50% post-thaw emergence was observed only in embryos with moisture contents below 20% (fresh weight) at developmental stages between 27 and 36 WAF, although more than 90% of  embryos could be dehydrated to < 20% moisture contents without loss in survival and  emergence as early as 21WAF. Nearly 80% embryos could be dehydrated safely to 20% moisture content as late as 45 WAF.

Keywords: cryopreservation, cryotolerance, desiccation tolerance, recalcitrant seed, seed development, palm.

 

 

CryoLetters 28 (4), 303-310 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

VITRIFICATION OF ZEBRAFISH EMBRYO BLASTOMERES IN MICROVOLUMES

J. Cardona-Costa* and F. García-Ximénez

Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Camino de Vera 14, 46071 Valencia, Spain.
Email:
cardona_costa_j@hotmail.com

Abstract

Cryopreservation of fish embryos may play an important role in biodiversity preservation and in aquaculture, but it is very difficult. In addition, the cryopreservation of fish embryo blastomeres makes conservation strategies feasible when they are used in germ-line chimaerism, including interspecific chimaerism.  Fish embryo blastomere cryopreservation has been achieved by equilibrium procedures, but to our knowledge, no data on vitrification procedures are available. In the present work, zebrafish embryo blastomeres were successfully vitrified in microvolumes: A number of 0.25 l drops, sufficient to contain all the blastomeres of an embryo at blastula stage (from 1000-cell stage to Oblong stage), were placed over a 2.5cm loop of nylon filament. In this procedure, where intracellular cryoprotectant permeation is not required, blastomeres were exposed to cryoprotectants for a maximum of 25 sec prior vitrification.  The assayed cryoprotectants (ethylene glycol, propylene glycol, dimethyl sulphoxide, glycerol and methanol) are all frequently used in fish embryo and blastomere cryopreservation.  Methanol was finally rejected because of the excessive concentration required for the vitrification (15M).  All other cryoprotectants were prepared (individually) at 5 M in Hanks' buffered salt solution (Sigma) plus 20% FBS (vitrification solutions: VS).
After direct thawing in Hanks' buffered salt solution plus 20% FBS, acceptable survival rates were obtained with ethylene glycol: 82.8%, propylene glycol: 87.7%, dimethyl sulphoxide: 93.4%, and glycerol: 73.9% (P<0.05).  Dimethyl sulphoxide showed the highest blastomere survival rate and allowed the rescue of as much as 20% of the total blastomeres from each zebrafish blastula embryo.

Keywords: Biodiversity, vitrification, microvolume, cryoprotectant, blastomeres, zebrafish.

 

 

CryoLetters 28 (4), 311-312 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

BOOK REVIEW

FREEZE DRYING OF PHARMACEUTICALS & BIOPHARMACEUTICALS, PRINCIPLES AND PRACTICE

by Felix Franks in association with Tony Auffret

Royal Society of Chemistry Publishing, Cambridge, UK. 2007 206pp ISBN 978-0-85404-268-5.

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