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CryoLetters 24 (5), 2003

CryoLetters 24, 269-274  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

SUBSTANCES WHICH INHIBIT ICE NUCLEATION: A REVIEW

C.B. Holt

Unilever R&D Colworth, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK.

Abstract

There are a number of substances described in the published literature which inhibit ice nucleation. Certain bacterial strains, mostly found among the nonfluorescent pseudomonade species, release material into the growth medium which reduces the nucleation temperature of water droplets to below that of distilled water. Extracts from the seeds of food crops including apricot, peach and plum can reduce the nucleation temperature of water droplets and dispersions of silver iodide. Antifreeze glycoproteins can reduce the nucleation temperature of saline solutions. Antifreeze proteins can inhibit the activity of some biological ice nucleators but not others. Certain novel polymers have been shown to inhibit the nucleation activity of dispersions of silver iodide and ice-nucleating bacteria.

Keywords: Supercooling, antifreeze proteins, freezing point

 

 

CryoLetters 24, 275-280  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ANTIOXIDATIVE PROPERTIES OF LACTOFERRIN FROM BOVINE COLOSTRUM BEFORE AND AFTER ITS LYOPHILIZATION

B.P. Sandomirsky*, S.E. Galchenko, K.S. Galchenko

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, 23 Pereyaslavska St., 61015 Kharkov, Ukraine.
E-mail:
cryo@online.kharkov.ua

Abstract

The effect of lactoferrin (LF) derived from native, frozen and lyophilized bovine colostrum on the intensity of free-radical processes in model systems has been investigated. It was shown that LF, not depending on the source of its obtaining, is an efficient iron chelator and decreases intensity of peroxidative processes. It was established, that antioxidative properties of LF from lyophilized colostrum have remained unchanged within 12 months of dry colostrum storage under proper conditions.

Keywords: bovine colostrum, lyophilization, lactoferrin, iron chelator, chemiluminescence, peroxidative processes.

 

 

CryoLetters 24, 281-292  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Desiccation and freezing tolerance of embryonic axes from Citrus sinensis [L.] OSB. pretreated with sucrose

Izulmé R. I. Santos1* and Cecil Stushnoff2

1 Embrapa Recursos Genéticos e Biotecnologia), CP 02372, CEP 70849-970, Brasília-DF, Brasil (corresponding author, e-mail izulme@cenargen.embrapa.br)
2 Colorado State University, Dept. of Horticulture and Landscape Architecture, Fort Collins, CO 80523, Colorado, USA.

Abstract

Embryonic axes of Citrus sinensis L. were successfully cryopreserved. While fully hydrated unfrozen axes germinated 100%, survival decreased as axes water content dropped, and total loss of viability was observed when the water content dropped to 0.04 and 0.10 mg H2O/mg dry mass, for axes without and with sucrose preculture, respectively. Fully hydrated axes did not survive exposure to liquid nitrogen. Highest seedling recovery (93-100%) for untreated axes was observed at 0.26 to 0.15 mg H2O/mg dry mass. Differential scanning calorimetry revealed the presence of broad melting peaks in fully hydrated embryonic axes. The size of the melting peak diminished as water was removed by desiccation. Minimum melting of water was observed at the point axes survived cryopreservation. Occurrence of a glass transition upon warming was not a condition for axes to survive liquid nitrogen exposure. In untreated axes, glucose, increased with desiccation to 0.2 mg H2O/mg dry mass, and decreased as the axes were desiccated to lower water contents. Fructose and sucrose levels did not increase when untreated samples were desiccated for the same periods of time. Raffinose and stachyose levels decreased as untreated and precultured embryonic axes were desiccated. In sucrose precultured axes, sucrose and fructose levels increased when they were dehydrated, reaching maximum levels at 0.2 mg H2O/mg dry mass. Tissue glucose did not change significantly with desiccation. Raffinose and stachyose levels dropped as precultured embryonic axes were dried.

Keywords: Cryopreservation, Citrus, embryonic axes, germplasm conservation, intermediate seed behaviour.

 

 

CryoLetters 24, 293-302  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECT OF BENZYLADENINE ON RECOVERY OF CRYOPRESERVED SHOOT TIPS OF GRAPEVINE AND CITRUS CULTURED IN VITRO

Qiaochun Wang, Ping Li, Özgur Batuman, Ron Gafny and Munir Mawassi*

Department of Virology, Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel.
*E-mail:
mawassi@volcani.agri.gov.il

Abstract

The effect of N6-benzyladenine (BA) on the recovery of cryopreserved shoot tips of the LN33 hybrid (Vitis L.) and 'Troyer' citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis {L.} Osbeck.] cultured in vitro was examined. For the LN33 hybrid, the presence of BA in the recovery medium was essential for survival of control and cryopreserved shoot tips, although the BA concentration did not influence the survival percentage. BA at 5, 2, and 5 ྒྷM or higher induced callus formation in control, and shoot tips cryopreserved by vitrification, and by encapsulation-dehydration, respectively. While a BA concentration of 4 ྒྷM was found optimal for recovery of control shoot tips, 1 and 2-4 ྒྷM produced the best recovery of shoot tips cryopreserved by vitrification and encapsulation-dehydration, respectively. A similar pattern of effect of BA on recovery was found for 'Troyer' citrange. Low survival of control and cryopreserved shoot tips was observed with a BA-free recovery medium. The addition of BA to the recovery medium significantly increased survival. The BA concentration that induced callus formation in shoot tips cryopreserved by encapsulation-vitrification was higher than that which induced it in those cryopreserved by encapsulation-dehydration. Recovery of control shoot tips was best with an addition of 6-10 ྒྷM BA to the medium. Optimal recovery of shoot tips cryopreserved by encapsulation-vitrification and encapsulation-dehydration was achieved with 3-4 and 2 ྒྷM BA, respectively. Results from the present study suggest that an optimal BA concentration for recovery of control shoot tips may be different from that for cryopreserved shoot tips; furthermore, the optimal BA concentration for recovery of cryopreserved shoot tips may also differ among different cryogenic procedures.

Keywords: Citrus, cryopreservation, dehydration, encapsulation, shoot tip, Vitis, vitrification.

 

 

CryoLetters 24, 303-314  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF MANGO (Mangifera indica L.) EMBRYOGENIC CULTURES

Yong-Jie Wu1, 2, Xue-Ling Huang1*, Jie-Ning Xiao1, Xiao-Ju Li1, Ming-De Zhou3 and Florent Engelmann4, 5

1The Key Laboratory of Gene Engineering of the Ministry of Education, Zhongshan University, Guangzhou 510275, China.
(*email:
ls17@zsu.edu.cn) (current address for Y.J. Wu).
2Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, 1 Guo Yan Road, Changli Town 066600, Hebei, China.
3IPGRI Subregional Office for East Asia, c/o CAAS, 12 Zhongguancun Nandajie, Beijing 100081, China.
4International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
5Cirad-Flhor, TA 50/PS4, Boulevard de la Lironde, 34398 Montpellier cedex 5, France (current address).

Abstract

Three techniques were compared for cryopreserving embryogenic masses (EMs) sampled from mango (Mangifera indica L.) cv. Zihua embryogenic cultures: (i) encapsulation/dehydration; (ii) pregrowth/dehydration; and (iii) vitrification. In all experiments, EMs were sampled from embryogenic cultures during their exponential growth phase and pretreated for 24 h on solid medium containing 0.5 M sucrose before freezing. No recovery was achieved after cryopreservation using the encapsulation/dehydration technique, whatever the moisture content (fresh weight basis) of EMs, which ranged from 78.3 % without dehydration to 40.8 % after 6 h dehydration. With the pregrowth/dehydration technique, limited recovery (8.3 %) was achieved after desiccation of EMs for 1 h, to 58.5 % MC. Using the vitrification technique, recovery ranged from 94.3 % after treatment of EMs with the PVS3 vitrification solution for 20 min (EM moisture content of 34.7 %) to 10.9 % after a 120 min treatment with the vitrification solution (EM moisture content of 26.0 %).

Keywords: Mangifera indica; embryogenic masses; vitrification; encapsulation/dehydration; pregrowth/dehydration.

 

 

CryoLetters 24, 315-322  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

GENETIC STABILITY ASSESSMENTS OF PLANTLETS REGENERATED FROM CRYOPRESERVED IN VITRO CULTURED GRAPE AND KIWI SHOOT-TIPS USING RAPD

Zhiyang Zhai1, Yongjie Wu1, Florent Engelmann3, Runzheng Chen1* and Yanhua Zhao2

1College of Life Science, Zhongshan University, Xinggang West Road 135, Guangzhou 510275, China (present address for Y. Wu).
2Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Hebei 066600, Changli Town, China.
3Institut de recherche pour le développement (IRD), BP 64501, 34394 Montpellier Cedex 5, France (present address).

Abstract

In vitro cultured shoot-tips of four grape (Vitis vinifera) cultivars and one kiwi (Actinidia deliciosa) cultivar were cryopreserved using the encapsulation-dehydration method. Genomic DNA of plantlets regenerated directly from cryopreserved shoot-tips was extracted and analyzed using the RAPD (Random Amplified Polymorphic DNA) technique. The RAPD profiles obtained were highly reproducible and no differences were found between the DNA patterns obtained with plantlets regenerated from control and cryopreserved plantlets. The RAPD technique therefore appears to be a fast, simple and efficient method for evaluating genetic stability of cryopreserved material, which can be used rapidly after the completion of a freezing experiment and will efficiently complement other genetic stability evaluation methods.

Keywords: grape, citrus, shoot-tip, cryopreservation, genetic stability, RAPD.

 

 

CryoLetters 24, 323-330  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

THE EFFECT OF ANTIFREEZE PROTEINS AND POLY(VINYL ALCOHOL) ON THE NUCLEATION OF ICE: A PRELIMINARY STUDY

C. B. Holt

Unilever R&D Colworth, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK
E-mail:
Chris.B.Holt@unilever.com

Abstract

     Three substances have been tested for ice nucleation inhibition. These were an antifreeze protein AFP III from the fish Macrozoarces americanus, an antifreeze glycoprotein AFGP from the fish Dissostichus mawsoni, and an 80% hydrolysed poly(vinyl alcohol) with a molecular weight of 9 to 10 kD. A nucleation spectrometer was used to test nucleation inhibition at a range of concentrations against two types of ice nuclei: those present in tap water and a bacterial nucleator from Pseudomonas syringae.  The PVA reduced the nucleation temperature of tap water and the bacterial dispersions at all the concentrations which were tested. The AFGP reduced the nucleation temperature of tap water but enhanced the nucleation activity of the bacterial nucleators. At low concentrations the AFP III reduced the nucleation temperature of both tap water and the bacterial nucleator. At high concentrations the AFP III enhanced the nucleation temperature of the bacterial nucleator and broadened the nucleation spectrum of the tap water to encompass the nucleation spread of the control. The possible mechanisms of nucleation suppression and enhancement are discussed.

Keywords: Supercooling, nucleation spectrometer.

 

 

CryoLetters 24, 331-340  (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

RAPID COLD HARDENING IN YOUNG HOPPERS OF THE MIGRATORY LOCUST Locusta migratoria L. (ORTHOPTERA: ACRIDIIDAE)

Xian-hui Wang, Le Kang*

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, the Chinese Academy of Sciences, Beijing, China 100080.
*E-mail:
lkang@panda.ioz.ac.cn

Abstract

This paper describes a rapid cold hardening process for first instar hoppers of the migratory locust Locusta migratoria L. First instar hoppers of this species are often subjected to subzero temperatures or frosts in early April or May after their emergence from the soil. The mean supercooling point of hoppers is –13.0 ± 1.4C; the fact that none could survive this temperature suggests they are freezing intolerant. When young hoppers were transferred directly from 30C to –7C for 2h, there was only 35.8% survival. However, exposure to 0C for 2h prior to transfer to –7C increased the apparent survival to 75%. A similar rapid cold hardening response can also be induced by gradual cooling at rates of between 0.05 and 0.1C min-1. Rapid cold hardening also elevates the Ltime50 of first instar hoppers at –7C by approximately 3 fold, and reduces the lethal temperature from –10C to –12C. However, the protection from cold shock gained through rapid cold hardening was transient and easily lost within 2h of hoppers being returned to 30C. The rapid cold hardening response is possibly advantageous to first instar hoppers that are often exposed to large temperature fluctuations in spring or early summer.

Keywords: Locusta migratoria, cold hardiness, acclimation, rapid cold hardening, cold shock

 

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