CryoLetters 28 (2), 69-76 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF EMBRYOGENIC TISSUES OF PINUS NIGRA ARN. BY A SLOW FREEZING METHOD

Terezia Salaj^1*, Bart Panis², Rony Swennen² and Jan Salaj¹

¹Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, PO Box 39A, 95007 Nitra, Slovak Republic.
*E-mail: nrgrtesa@savba.sk
²Laboratory of Tropical Crop Improvement, K.U. Leuven, Kasteelpark Arenberg 13, B-3001 Leuven, Belgium

Abstract

Six different embryogenic cell lines of Pinus nigra Arn. have been cryopreserved in liquid nitrogen using cryoprotection with sucrose (18%) and DMSO (7.5%). Post-thaw growth and tissue proliferation have been observed in five cell lines. The survival levels after storage in liquid nitrogen reached values between 62.5 and 100%. Growth of recovered embryogenic cells as well as somatic embryos is similar to the non-frozen tissues maintained in long-term culture. Somatic embryo maturation and plantlet regeneration occurred in all selected cell lines.

Keywords: cryopreservation, embryogenic tissues, Pinus nigra, plantlet regeneration

CryoLetters 28 (2), 77-81 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

LARCH (LARIX KAEMPFERI) XYLEM PARENCHYMA CELLS RESPOND TO SUBFREEZING TEMPERATURE BY DEEP SUPERCOOLING

Jun Kasuga¹, Naoki Takata¹, Kenichi Yamane¹, Katsushi Kuroda²,
Keita Arakawa¹ and Seizo Fujikawa¹*

¹Laboratory of Woody Plant Biology, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.
²Department of Wood Properties, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, 305-8687 Japan.
*Corresponding author: sfuji@for.agr.hokudai.ac.jp

Abstract

In previous studies, xylem parenchyma cells (XPCs) in the boreal softwood species larch, which has thick and rigid walls similar to those of XPCs in boreal hardwood species, were shown to respond to subfreezing temperature by deep supercooling during summer but change their freezing behavior to extracellular freezing during winter (4). In this study, we re-examined freezing behavior of XPCs in larch by observation of deep etching of frozen samples as well as observation of re-warmed samples after freezing using a cryo-scanning electron microscope. The results showed that XPCs in larch adapts to subfreezing temperature by deep supercooling throughout all seasons.  Such freezing behavior is the same as that of XPCs in boreal hardwood species.

Keywords: xylem parenchyma cells (XPCs), deep supercooling, cryo-scanning electron microscope (cryo-SEM), larch (Larix kaempferi)

CryoLetters 28 (2), 83-94 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

CRYOPRESERVATION OF PISTACIA SPP. SEEDS BY DEHYDRATION AND ONE-STEP FREEZING

Y. Ozden-Tokatli¹, E.A. Ozudogru¹, F. Gumusel¹, M. Lambardi ²*

¹ Gebze Institute of Technology, Faculty of Science, Department of Biology. Istanbul cad. No: 101, 41400, Gebze, Kocaeli, Turkey
² IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR/Consiglio Nazionale delle Ricerche, Polo Scientifico, 50019 Sesto Fiorentino (Firenze), Italy
* For correspondence: lambardi@ivalsa.cnr.it

Abstract

Cryopreservation protocols by dehydration and one-step freezing were developed for seeds from three Pistacia species, i.e., P. vera, P. terebinthus and P. lentiscus, which were characterised by different initial germination percentages (100%, 17% and 81%, respectively). In P. vera, a maximum of 90% germination was obtained following 8 hours drying in silica gel (corresponding to 11.7% moisture content on a FW basis) and direct immersion in LN.  In P. terebinthus and P. lentiscus, shorter periods of dehydration (1 hour and 15 min, respectively) were sufficient to reduce their moisture content to about 20%, which resulted in peak seed germination percentages from cryostorage of 16% and 47%, respectively. Following cryopreservation, the seeds germinated better on semi-solid MS medium, than on cotton wool wetted with dH₂O or liquid MS medium. Finally, in P. vera and P. lentiscus, high and significant correlation coefficients were obtained between the TTC viability test and seed germinability after recovery from LN, provided that seeds which were considered positive in the test showed completely or partially red embryonic axes coupled to completely red cotyledons.   

Keywords: Pistacia, cryopreservation, seed, dehydration, triphenyl tetrazolium chloride (TTC)

CryoLetters 28 (2), 95-108 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

INTRACELLULAR SUGARS IMPROVE SURVIVAL OF HUMAN RED BLOOD CELLS CRYOPRESERVED AT -80ºC IN THE PRESENCE OF POLYVINYL PYRROLIDONE AND HUMAN SERUM ALBUMIN

GuoBo Quan^1*, Liang Zhang¹, Yong Guo¹, MinXia Liu, JieXi Wang¹, Yan Wang¹,
Bo Dong², An Liu, JinGang Zhang¹, Ying Han^1*

¹Institute of Transfusion Medicine, Beijing 100850, China;
²Institute of Radiation Medicine, Beijing 100850, China.
E-mail: hanying1001@yahoo.com.cn, waltq20020109@yahoo.com.cn

Abstract

Cryopreservation with impermeable protectants has great significance on storage of human red blood cells. It has become feasible to use glycerol free cryopreservation for human red blood cells. This study focuses on the effect of intracellular trehalose or glucose on human red blood cells cryopreserved in the presence of polymer. Red blood cells were cryopreserved for 48h-72 h at -80ºC. The data showed that the loading efficiency of glucose was significantly higher than that of trehalose, but trehalose loading process induced more hemolysis than glucose loading process. Compared with the other groups, the combination of intracellular glucose, PVP, and human serum albumin can significantly decrease the percent hemolysis after cryopreservation (P<0.01). However, the percent hemolysis induced by intracellular trehalose was less than that induced by extracellular trehalose, but the difference was not significant (P>0.05). The adenosine 5'-triphosphate (ATP) level and 2,3-diphosphoglycerate (2,3-DPG) level of cryopreserved red blood cells were significantly less than those of fresh red blood cells. However, sugars can provide certain protection for ATP and 2, 3-DPG compared with red blood cells cryopreserved in the absence of sugars. The protection of glucose on the metabolic function was more than that of trehalose. Cryopreservation can increase the percentage of cells with exposed phosphatidylserine (PS), but the ability of trehalose to maintain PS normal distribution is higher than that of glucose. Furthermore, intracellular sugars can protect membrane integrity of cryopreserved red blood cells, although a small portion of cells appeared spherocytic or echinocytic shape. Finally, most membrane proteins of cryopreserved red blood cells were similar to the membrane proteins of fresh red blood cells, but trehalose can result in loss of glyceraldehyde phosphate dehydrogenase (GAPD) and peroxiredoxin 2. In conclusion, it is feasible to cryopreserve red blood cells using polymer, human albumin and sugars as main protectants. The cryoprotective effect of glucose may be better than that of trehalose in the presence of PVP and human serum albumin, because sugar loading process causes more cell injuries in case of trehalose compared to glucose, and these injuries in turn manifest themselves during subsequent cryopreservation and thawing. In the future, finding an approach to decrease the injuries during trehalose loading process still is critical.

Keywords: Human red blood cell, Cryopreservation, Trehalose, Glucose, Polyvinyl pyrrolidone, Human serum albumin

CryoLetters 28 (2), 109-118 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

ACQUISITION OF CRYOTOLERANCE IN MAIZE EMBRYOS DURING SEED DEVELOPMENT

Bin Wen^* and Songquan Song

Xishuangbanna Tropical Botanical Garden, the Chinese Academy of Sciences, Mengla, Yunnan, China, 666303
^*To whom correspondence should be addressed (E-mail: wenb@xtbg.org.cn).

Abstract

A desiccation-based cryopreservation protocol was employed to study the development of cryotolerance and desiccation tolerance in maize embryos from 23 to 50 days after pollination (DAP). Tolerances were acquired gradually and concomitantly. Maize embryos had low desiccation tolerance at 23 DAP when assessed by survival (embryo elongation) and emergence (root and shoot growth) after dehydration. Desiccation tolerance increased progressively, reached its maximum at 38 DAP, and remained constant afterwards. Cryotolerance, assessed by survival and emergence of post-thaw embryos, however, was nil until 26 DAP. Embryos at 29 DAP withstood cryoexposure within a very narrow moisture range only. Throughout development cryotolerance increased gradually, reached a maximum at 44 DAP and then remained at this level. The correlation between moisture content and cryopreservation success was notably influenced by the maize embryo's development stage. As seeds developed, the moisture content allowing 90% dehydrated embryos to survive and to emerge decreased, while the upper moisture content allowing 50% post-thaw embryos to survive and to emerge increased. Moisture contents of c. 14% allowed no less than 50% post-thaw embryos to emerge at the later development stages (e.g. c. 44 DAP); but no embryos within the same moisture range survived cryoexposure at 29 DAP, although they could withstand desiccation to this moisture level without impairment of survival and emergence. The relationship between cryotolerance and desiccation tolerance during maize seed development is discussed.

Keywords: cryopreservation; cryotolerance; desiccation tolerance; orthodox seeds; recalcitrant seeds; seed development; Zea mays L.

CryoLetters 28 (2), 119-128 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

NEW CALORIMETRIC SYSTEM AND SOME RESULTS OF WATER PHASE TRANSITION RESEARCH IN PLANT ROOTS

N. Bakradze*, E. Kiziria, V. Sokhadze, Sh. Gogichaishvili, and E. Vardidze

E. Andronikashvili Institute of Physics, 6 Tamarashvili str, Tbilisi, 0107 Georgia
* Email: nugzarb@yahoo.com

Abstract

The principle of operation and main parameters of the recently created scanning differential reverse microcalorimeter of the new generation are presented. The microcalorimeter is destined for studying water crystallization and ice melting processes in heterogeneous systems, including plant and animal cells and tissues in the temperature range of 20 to –55°С. In order to obtain maximum information from the experimental results respective algorithms and applied software package were developed. The results of studies of water crystallization and ice melting processes in different parts of common plantain (Plantago major L.) root, as a certain model system, can give us information on the peculiarities of the studied processes in complex, heterogeneous systems.

Keywords: DSC, plant root, water supercooling, intracellular crystallization.

CryoLetters 28 (2), 129-136 (2007)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

EFFECTS OF REMOVALOF NECROTIC BLASTOMERES FROM HUMAN CRYOPRESERVED EMBRYOS ON PREGNANCY OUTCOME

Wei-Xin Liu^{1, 2}, Meng-Jun Luo², Ping Huang², Li Wang²,Cheng-Yuan Zhao², Li-Min Yue¹ and Yu Zheng¹*

¹ Department of Physiology, Sichuan University, Chengdu, 610041, China
² Chengdu Institute of Family Planning, Chengdu, 610031, China
* Corresponding author: E-mail: yzheng@wcums.edu.cn

Abstract

This study assessed whether the implantation potential of embryos that were partially damaged after freezing and thawing can be improved by removal of necrotic blastomeres. We retrospectively analyzed the pregnancy rate and implantation rate of 170 human frozen embryo transfer cycles. Laser-assisted hatching and micromanipulation were performed to remove the necrotic blastomeres. A higher clinical pregnancy rate (22.22%) and embryo implantation rate (10.17%) were observed when transferred embryos comprised fully intact and partially damaged embryos compared with partially damaged embryos alone (5.88% and 2.82%, respectively). When transferred embryos were fully intact and partially damaged embryos, removal of necrotic blastomeres from partially damaged embryos significantly increased the clinical pregnancy rate (43.90% versus 24.00%, P<0.05) and the implantation rate (19.44% versus 10.29%, P<0.05). The results indicated that the implantation potential of partially damaged cryopreserved embryos can be improved by removal of necrotic blastomeres with laser-assisted hatching and micromanipulation.

Keywords: Human embryo; Cryopreservation, necrotic blastomeres, implantation potential.

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