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Abstracts: CryoLetters 28 (1), 2007

CryoLetters 28 (1), 1-12 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF COLD-ACCLIMATED MINT (MENTHA SPP.) SHOOT TIPS USING A SIMPLE VITRIFICATION PROTOCOL

Angelika Senula1, E.R. Joachim Keller*1, Tekshbayar Sanduijav2
and Tamene Yohannes3

1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany.
E-mail:
keller@ipk-gatersleben.de
2 Mongolian State University of Agriculture, Zaisan-210153, Ulaanbaatar, Mongolia
3 Institute of Biodiversity Conservation and Research (IBCR), P. O. Box 30726, Addis Ababa, Ethiopia

Abstract

Accessions of Mentha x piperita, M. x villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant re-growth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25°C or in alternating temperature regimes (25 / 15°C or 25 / -1°C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25°C / -1°C than in plants cultivated at 20 or 25°C or at 25°C / 15°C for all nine tested accessions. The mean re-growth levels increased from 36% at 20 °C to 69% at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89%. A pre-culture at alternating temperatures of 25°C / 15°C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.

Keywords: Mint, shoot tips, vitrification, PVS 2, aluminium foil, cold acclimation, loading solution

 

 

CryoLetters 28 (1), 13-22 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF PEACH PALM ZYGOTIC EMBRYOS

Douglas A. Steinmacher1,2, Cleber W. Saldanha3, Charles R. Clement4
and Miguel P. Guerra1*

1UFSC, Universidade Federal de Santa Catarina, Dept Fitotecnia/LFDGV, 88040-900 Florianópolis, SC, Brazil
2Present address: Department of Crop Science and Plant Ecology, Biocentre Klein Flottbek and Botanical Garden, Ohnhorststr. 18, 22609 Hamburg, Germany.
3UFSM, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.
4INPA, Instituto Nacional de Pesquisas da Amazônia, Cx Postal 478, 69011-970 Manaus, AM, Brazil
*Author for contact
mpguerra@cca.ufsc.br

Abstract

Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3% sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83% and 87% to 18% and 20% for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20% water content, followed by rapid freezing in liquid nitrogen (-196°C) and rapid thawing at 45°C. In these conditions 29% of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41±9% and 88±4% survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.

Keywords: Bactris gasipaes, germplasm conservation, in vitro culture, acclimatization

 

 

CryoLetters 28 (1), 23-32 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF IN VITRO SHOOT APICES OF OXALIS TUBEROSA MOL.

M. E. González-Benito1*, V. H. Mendoza-Condori1,2 and A.D. Molina-García3

1 Dept. Biología Vegetal, Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad Politécnica de Madrid, Madrid 28040, Spain.
2 Present address: División de Agricultura, Instituto Boliviano de Ciencia y Tecnologia Nuclear (IBTEN) Av. 6 de Agosto 2905, Casilla 4821 - La Paz, Bolivia.
3 Instituto del Frío, CSIC, José Antonio Novais 10, Ciudad Universitaria, Madrid 28040, Spain.
*corresponding author:
me.gonzalezbenito@upm.es

Abstract

Oca (Oxalis tuberosa Mol.) is an under-utilized tuber crop from the Andean region. Cryopreservation would allow the safe and long-term preservation of the genetic resources of this crop. A protocol for the cryopreservation of in vitro grown shoots has been developed using the vitrification solution PVS2. Two genotypes were studied (G1 and G27). Nodal segments were cultured on MS medium and incubated at 10ºC with 16 h photoperiod and 10 μmol m-2s-1 irradiance, for two weeks. Apices were then excised and cultured on MS +     0.15 M sucrose for 3 days at 5ºC in darkness. Subsequently, apices were immersed in a loading solution (liquid MS medium + 2 M glycerol + 0.4 M sucrose), and then treated with the vitrification solution PVS2 for 0-40 minutes. Cryovials were then immersed in liquid nitrogen. Four weeks after rewarming and culture on recovery medium, genotype G1 showed approximately 60% recovery (normal growth) with 20 min PVS2 treatment. Genotype G27 showed lower recovery (30%). Differential scanning calorimetry yielded a Tg midpoint for PSV2 solution of ca. -120ºC. Calorimetric studies on apices at different stages of the cryopreservation protocol showed a change in calorimetric parameters consistent with a decrease in the amount of frozen water as the protocol advanced.

Keywords: Andean tubers, calorimetry, cryopreservation, oca, rapid cooling, vitrification

 

 

CryoLetters 28 (1), 33-37 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

INCREASED DIETARY CHOLESTEROL ENHANCES COLD TOLERANCE IN DROSOPHILA MELANOGASTER

Scott M. Shreve, Shu-Xia Yi and Richard E. Lee, Jr.*

*Department of Zoology, Miami University, Oxford, OH 45056;
E-mail:
leere@muohio.edu

Abstract

For many years, non-freezing cold shock injury has been associated with damage to the cell membrane.  In this study, we enhanced membrane cholesterol levels of Drosophila melanogaster by raising larvae on a cholesterol-augmented diet. Diet augmentation significantly increased the amount of cholesterol in the cell membranes of the adult flies (1.57 ± 0.17 nmol mg-1 vs. 0.93 ± 0.11 nmol mg-1). Flies on the cholesterol-augmented diet exhibited a greater intrinsic cold tolerance: this group had a higher survival rate after a 2-h cold shock of -5°C than did the control group (71.0 ± 6.6% vs. 36.0 ± 8.1%). Cholesterol-augmented flies also had a significantly greater capacity to rapidly cold-harden to -7°C (36.7 ± 4.4%) compared to flies on a control diet (20.0 ± 2.9%). These results suggest a mechanistic link between protection from chilling or cold shock injury and modifications to the cellular membrane.

Keywords: cholesterol, membrane, insect cold tolerance, cold shock

 

 

CryoLetters 28 (1), 39-49 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A NOVEL, INTRACELLULAR ANTIFREEZE PROTEIN IN AN ANTARCTIC BACTERIUM, FLAVOBACTERIUM XANTHUM

Hidehisa Kawahara1*, Yoshiko Iwanaka1, Sakura Higa1, Naomi Muryoi1,
Mika Sato2, Michinori Honda2, Hironori Omura2 and Hitoshi Obata1

1Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan,
2Ikeda Food Research Co., Ltd., 95-7 Sasaoki-cho, Fukuyama-shi, Hiroshima 721-0956, Japan.
E-mail:
kawahara@ipcku.kansai-u.ac.jp

Abstract

One strain of Antarctic bacteria, Flavobacterium xanthum IAM12026, has a highly active antifreeze protein (AFP) in the intracellular space. The cell-free extract from strain IAM12026 after culturing at 4°C for 7 days in TSB medium, had activity of 0.04°C at a concentration of 0.7 mg/ml. The ice crystals formed do not have distinct facets without typically rounded shape and the changes of their morphology during the course of the thermal hysteresis (TH) measurement. The ice crystal 'burst' occurring at the end-point of the TH is dendritic with hexagonal symmetry. Also, this activity was not affected by the treatment of dialysis and the addition of EDTA. Furthermore, this cell-free extract had high levels of ice recrystallization-inhibiting (RI) activity like those of Fish AFPs. The AFP (FlAFP) was homogeneity purified using chromatography. A relative molecular mass of approximately 59,000 was calculated from gel filtration and SDS-PAGE data. The thermal stability of FlAFP was below 50°C, and TH value was absent above 60°C. The TH value of FlAFP was activated at 5.2°C by the addition of 0.5 M malate. This activation was decreased with increasing protein concentration. To our knowledge this is the first report on the high level of TH and RI activities of bacterial intracellular AFP.

Keywords: Flavobacterium xanthum IAM12026; antifreeze protein; thermal hysteresis; ice recrystallization-inhibiting activity

 

 

CryoLetters 28 (1), 51-60 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

PURIFICATION AND STRUCTURAL ANALYSIS OF A TYPE III ANTIFREEZE PROTEIN FROM THE EUROPEAN EELPOUT ZOARCES VIVIPARUS

C.N. Albers, M. Bjørn-Mortensen, P.E. Hansen, H. Ramløv and T.F. Sørensen*

Dept. Life Sciences and Chemistry, Roskilde University, P.O. Box 260, 4000 Roskilde, DK
* Corresponding author:
tfs@ruc.dk

Abstract

It has recently been reported that the eelpout Zoarces viviparus synthesizes a family of antifreeze proteins (AFP) similar in sequence to type III AFPs. A method has been set up to separate these antifreeze proteins from blood serum of this teleost species. A total of nine proteins with antifreeze activity have been isolated, several to a purity suited for NMR experiments. One of the proteins, Zvafp13, has been subject to partial structure determination by NMR. 1D- and 2D-1H NMR analyses were carried out. In the 1D-experiments it was observed that the protein contained 28 slow-exchanging amides, suggesting a compact structure. The 2D-experiments were utilized to assign observed signals to specific amino acids. From TOCSY- and NOESY-experiments 35 out of a total of 66 amino acids were assigned. The amide exchange pattern, protein primary sequence, chemical shifts and NOE-cross-peaks between amides and α-protons in the β-sheets suggest that Zvafp13 structurally resembles the recombinant type III AFP rQAE m1.1.

Keywords: Zoarces viviparus, type III AFP, protein purification, 2D-NMR

 

 

CryoLetters 28 (1), 61-68 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

RECRYSTALIZATION INHIBITION ASSESSED BY SPLAT COOLING AND OPTICAL RECRYSTALOMETRY

David A. Wharton1*, Peter W. Wilson2, Jodi S. Mutch3, Craig J. Marshall3 and Miang Lim4

Departments of Zoology1, Physics2, Biochemistry3 and Food Science4, University of Otago, P.O. Box 56, Dunedin, New Zealand
*Corresponding author 
david.wharton@stonebow.otago.ac.nz

Abstract

Levels of recrystalization have been measured by two distinct techniques; a splat cooling assay and a new device, an optical recrystalometer, which measures the change in light transmittance through a frozen sample. Both techniques indicate the presence of recrystalization inhibitors in a grass extract and in other samples. The advantages of each method of measuring recrystalization are discussed.

Keywords: ice, recrystalization, splat freezing, antifreeze protein

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