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ABSTRACT ARCHIVE |
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CryoLetters is a bimonthly, international journal for low temperature science and technology |
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CryoLetters 32 (6), 447-450 (2011) FREEZING EFFECT ON CHIRALITY AMPLIFICATION OF L-ASPARAGINE BY CRYSTALLIZATION OF THE RACEMATE IN PREFERENTIAL CONDITION Tamás Vajda* and Miklós Hollósi Laboratory of Structural Chemistry and Biology, Department of Organic Chemistry, Institute of Chemistry, Eötvös Loránd University, P.O.Box 32, Budapest 112, H-1518, Hungary; Fax: +36-1-372-2620; Abstract The role of freezing in stereoselection by crystallization of a saturated DL-asparagine solution in preferential condition of L-asparagine, has been investigated. To this end, the L-asparagine excess obtained by crystallization and then kept of different lengths of time at 20oC in frozen aqueous solution was analyzed. The samples of the yielded materials were derivatised according to Marfey’s method (see text). These derivatives were traced and evaluated by RP-HPLC analysis. The relatively best effects appeared after a one day treatment, where the freezing induced asymmetry amplifications and it increased the L-enantiomer excess from 10 mol % to 34.9, 35.0 and 35.5 mol %, respectively. Here we provide the first example of the influence of freezing on chirality amplification by preferential crystallization. Also some speculations are made about the implications of our findings in the prebiotic events of L-amino acids. Keywords: freezing, crystallization, asparagine, chirality amplification, prebiotic amino acids.
CryoLetters 32 (6), 451-462 (2011) PALM CRYOBANKING Lotfi Fki1*, Neila Bouaziz1, Nahed Sahnoun1, Rony Swennen2, 3, Noureddine Drira1 and Bart Panis2 1Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, University of Sfax, Route Sokra BP 1171, 3000 Sfax, Tunisia. Abstract We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety ‘Barhee’. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg.L-1 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3% when PEMs were treated with PVS2 for 30 min at 0°C and 56.7% when PVS2 treatment lasted for 15 min at 25°C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 ± 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation. Keywords: Cryobank, dates, droplet-vitrification, plant genetic resources, somaclonal variation, tissue culture.
CryoLetters 32 (6), 463-472 (2011) CRYOPRESERVATION OF SHOOT TIPS AND COTYLEDONS OF THE NORTH AMERICAN GINSENG (Panax quinquefolius L.) E.E.Uchendu1*, D.C.W. Brown2 and P.K. Saxena1* 1Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Abstract North American ginseng (NAG) (Panax quinqueolius L.) is a medicinal plant in high demand due to its health benefits. Cryopreservation is a good alternative for long-term conservation of NAG germplasm. Pretreatments of shoot tips (0.8-1 mm) and cotyledons (1-2 mm) on sucrose and abscisic acid (ABA) enriched medium were tested to determine the effects on regrowth following cryopreservation in liquid nitrogen. The maximum regrowth (60%) following PVS2 vitrification occurred with shoot tips after three weeks of cold acclimation and pretreatment on sucrose (0.3 M) or a combination of ABA (0.1 M) and sucrose in the 3rd week. Cotyledon recovery was best with the combination pretreatment. Shoot tips showed normal development and cotyledons produced embryogenic callus after the cryopreservation process. This is the first report on cryopreservation of shoot tips and cotyledons of Panax species. This cryopreservation protocol provides a safe long-term storage method for important NAG selections and makes it possible to use cryopreservation for improving the security of NAG germplasm. Keywords: abscisic acid (ABA), medicinal plant, pretreatment, somatic embryogenesis, sucrose and vitrification
CryoLetters 32 (6), 473-476 (2011) SEMINAL FREEZING IN Pure Breed Andalusian horse: DIFFERENCE IN INDIVIDUAL STALLIONS AND CORRELATION BETWEEN PRE AND POST-FREEZING SPERM PARAMETERS Ruiz, L.1, Echegaray, A.2*and Lafuente, A.1 1 C.I.F.E.A. of Lorca (Murcia). Consejería de Agricultura y Agua, Murcia, Spain. Abstract The aim of this study was the optimization of the sperm freezing protocols for the Pure Breed Andalusian Horse (AH) stallions. The study was performed in 84 ejaculates from 14 stallions (6 ejaculates per stallion). We examined the effect of individual stallion, centrifugal force and centrifugation extender on post-thaw sperm quality. Neither centrifugal force nor centrifugal extender had any significant effect on post-centrifugation or post-thawing sperm quality. Stallion was the principal source of variation in our experiments, showing individual significant differences (p<0.05) in all parameters. Individual differences were more extreme prior to freezing than after freezing-thawing. There are significant positive correlations (p<0.05 and p<0.01) between all post-centrifugation and post-thawing parameters. Keywords: Sperm, centrifugation, stallion, freezing, Andalusian Purebred Horse
CryoLetters 32 (6), 477-486 (2011) GENETICALLLY ENGINEREED TREHALOSE ACCUMULATION IMPROVES CRYOPRESERVATION TOLERANCE OF CHRYSANTHEMUM (Dendranthema grandiflorum KITAM.) SHOOT-TIPS Antelmo Osorio-Saenz1, Jose Oscar Mascorro-Gallardo1*, María del Rocío Valle-Sandoval1, María Teresa González-Arnao2* and Florent Engelmann3, 4 1Universidad Autónoma Chapingo, Departamento de Fitotecnia, Chapingo Estado de México, México. *E-mail: joscar@correo.chapingo.mx Abstract Shoot-tips isolated from two transgenic lines of chrysanthemum (Dendranthema grandiflorum Kitam.) var. Indianapolis in vitro plantlets with induced capacity to biosynthesize trehalose, and from a non-transformed line, were subjected to cryopreservation using a vitrification procedure. After dissection, apices were precultured on semi-solid MS medium with 0.3 M sucrose for 4 days, loaded in a 0.4 M sucrose + 2 M glycerol solution for 20-30 min and exposed to PVS2 or PVS3 vitrification solutions for 0, 20, 40 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. The highest shoot regeneration after cryopreservation was obtained with exposure to either PVS solution for 40 min. Plant regeneration from cryopreserved shoot-tips ranged between 48% and 67% for transgenic lines and between 33% and 36% for non-transgenic lines. No polymorphic loci were detected in plantlets regenerated from cryopreserved and non-cryopreserved shoot-tips with RAPD techniques using eight primers that amplified 101 monomorphic loci. Keywords: chrysanthemum, trehalose, transgenic plantlets, shoot-tips, vitrification
CryoLetters 32 (6), 486-497 (2011) IMPROVED CRYOPRESERVATION OF CHRYSANTHEMUM (Chrysanthemum morifolium) USING DROPLET-VITRIFICATION Yoon-Geol Lee1,4, Elena Popova1, 2, Hai-Yan Cui3, Haeng-Hoon Kim1*, Sang-Un Park4, Chang-Hyu Bae5, Sheong-Chun Lee5 and Florent Engelmann6, 7 1National Agrobiodiversity Center, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea. Abstract A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.3, 0.5 and 0.7 M for 31,17 and 7 h, respectively). Precultured explants were treated for 40 min with C4 loading solution comprising (w/v) 17.5% glycerol + 17.5% sucrose, then dehydrated with PVS3 vitrification solution (w/v, 50% glycerol + 50% sucrose) for 60 min (axillary shoot tips) or 90 min (apical shoots). Explants were cryopreserved by direct immersion in liquid nitrogen in minute drops of PVS3 attached to aluminum foil strips. The optimal age of donor plants was 4-5.5 weeks for apical shoots and 7 weeks for axillary shoot tips, producing post-cryopreservation regeneration percentages of 81.9% and 84.9%, respectively. Plants regenerated from cryopreserved samples showed no phenotypical abnormalities and similar profiles of relative DNA content were recorded for control and cryopreserved plants. Our results suggest that the modified droplet-vitrification protocol described in this paper is highly effective and may prove user-friendlier than the cryopreservation protocols already published for chrysanthemum. Keywords: loading, preculture, relative DNA content, shoot tips, vitrification solution.
CryoLetters 32 (6), 498-505 (2011) LONG-TERM CONSERVATION THROUGH CRYOPRESERVATION OF IMMATURE SEED OF Mantisia spathulata AND Mantisia wengeri; TWO ENDANGERED PLANTS OF NORTH-EAST INDIA S. S. Das Bhowmik1*, S. Kumaria2 and P. Tandon2 1Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, 781039 India Abstract A successful protocol for long-term conservation of two endangered plants viz. Mantisia spathulata and M. wengeri has been devised through cryopreservation of immature seeds. Immature seeds of both the species were precultured in 0.6 M Sucrose and 2 M Glycerol for 3 h at 24 ± 2ºC. Precultured seeds were then desiccated under the airflow of 27 ± 3 m min -1 velocity inside laminar air flow cabinet for different time periods. The seeds were then cryostored in liquid nitrogen for an hour. A maximum of 40% and 36.6% seed germination was recorded after cryostorage at moisture contents of 26.0% and 16.2% for M. spathulata and M. wengeri respectively. To protect these rare plants against loss due to disease, insect damage, or natural disaster a back up collection has been established using the protocol and applied to a large number of immature seeds that were obtained from the ex situ plants growing in the experimental garden of the North-eastern Hill University, Shillong. Keywords: long-term conservation, cryopreservation, germination, moisture content, dehydration
CryoLetters 32 (6), 506-515 (2011) EEFFECT OF PRECULTURE, PVS2 AND VITAMIN C ON SURVIVAL OF RECALCITRANT Nephelium ramboutan-ake SHOOT TIPS AFTER CRYOPRESERVATION BY VITRIFICATION S.P. Chua1 and M.N. Normah2* 1School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. Abstract This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0°C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25°C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3%. It was only 53.3% when shoot tips were exposed to PVS2 at 25°C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.67%) without LN exposure, compared to 73.3% for shoot tips not treated with vitamin C. Moreover, 3.3% shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species. Keywords: cryopreservation, preculture, liquid nitrogen, Nephelium ramboutan-ake, shoot tips, vitamin C, vitrification.
CryoLetters 32 (6), 516-524 (2011) FREEZE-THAWING SINGLE HUMAN EMBRYONIC STEM CELLS INDUCE E-CADHERIN AND ACTIN FILAMENT NETWORK DISRUPTION VIA G13 SIGNALING Hinako Ichikawa1, Susumu Yoshie1, Sakiko Shirasawa2, Tadayuki Yokoyama2, Fengming Yue1, Daihachiro Tomotsune1, and Katsunori Sasaki1* 1Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Abstract Poor adhesion of single human embryonic stem (hES) cells after freeze-thawing causes death. To investigate mechanisms responsible for this, Rho-dependent protein kinase (ROCK) inhibitor Y-27632-treated and untreated single hES cells were analyzed for E-cadherin and F-actin distribution by immunostaining and phalloidin staining respectively and for G13 signaling pathway components by DNA microarray and quantitative polymerase chain reaction (PCR). Y-27632-treated cells clustered rapidly and maintained E-cadherin and F-actin distribution without losing Oct3/4. Immediately after thawing, E-cadherin in untreated hES cells dotted along the membrane and then displayed eccentric cytoplasmic localization. Bleb formation and early Oct3/4 loss occurred after F-actin network condensation in the cytoplasm. Microarray analyses and quantitative PCR indicated upregulation of two actin reorganization-associated components of the G13 signaling pathway, Arhgdib and Cdc42, in untreated cells. Considering these findings and that cell death was partly interrupted by Y-27632, E-cadherin and actin cytoskeleton network disruption through the G13 signaling pathway may cause hES cell death after freeze-thawing. Keywords: Cryopreservation, E-cadherin, actin filaments, G13 signaling pathway, human embryonic stem cells, ROCK inhibitor
CryoLetters 32 (6), 525-536 (2011) IMPACT OF CRYOPROTECTANTS AND CRYOPRESERVATION ON METABOLIC ACTIVITY AND CYTOSKELETON PROTEINS OF ZEBRAFISH (Danio rerio) OVARIAN FRAGMENTS Tiziana Zampolla, Emma Spikings, Srimathuri Srirangarajah, David M. Rawson and Tiantian Zhang* LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK Abstract Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution. Keywords: zebrafish; ovarian fragments; F-actin; tubulin; cytoskeleton; mitochondria; cryopreservation; methanol. |
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For Abstracts published from meetings, such as SLTB meetings, go to the
relevant Volume Year of the journal (above). For Full text Free Access Content (from 2000 onwards) go to CryoLetters at Ingenta and look for the blue symbol. |
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