Volume 35, No. 3 May/June 2014 ISSN 0143-2044
	Relative importance of seed drying rate, desiccation tolerance, and cryotolerance for the conservation of Ardisia elliptica, A. brunnescens and A. virens Xin Yao, Uromi Manage Goodale, Zhilin Li, Y. Huang, X. F. Wang, F. Y. Cheng, Y. H. Tan, C. F. Xiao, and Qinying Lan	162-170
	Relation between frost tolerance and post-cryogenic recovery in Hypericum SPP Linda Petijová, Katarína Bruňáková, Jiří Zámečník, Daniela Zubrická,Anna Mišianiková, Katarína Kimáková and Eva Čellárová	171-179
	Use of fm1-43, a membrane-specific fluorescent dye, to estimate plasma membrane integrity in the cryopreservation of green algae Tomokazu Yamazaki, Chizuru Hirai, Shuhei Ota, Kazuyoshi Kuwano, and Shigeyuki Kawano	180-187
	Cryopreservation of quince (Cydonia oblonga Mill.) Paul T. Lynch, Ayesha Siddika, Aradhana Mehra, Carla Benelli and Maurizio Lambardi	188-196
	Growth medium alterations improve in vitro cold storage of pear germplasm Irina Kovalchuk, Zhangul Zhumagulovа, Timur Turdiev and Barbara M. Reed	197-203
	Cryopreservation of Passiflora pohlii nodal segments and assessment of genetic stability of regenerated plants T. S. M. Merhy, M.G. Vianna, R.O. Garcia, G. Pacheco and E. Mansur	204-215
	Phenotypic and molecular characterization of plants regenerated from noncryopreserved and cryopreserved wild Solanum lycopersicum MILL. Seeds Byron Zevallos, Inaudis Cejas, Florent Engelmann, Domenico Carputo, Riccardo Aversano, Maria-Teresa Scarano, Ermis Yanes, Marcos Martínez-Montero and José Carlos Lorenzo	216-225
	NIV versus dropping vitrification in cryopreservation of human ovarian tissue Zhun Xiao, Shang–Wei Li, Yao-Yao Zhang, Yan Wang, Ling-ling Li and Wei Fan	226-231
	Developmental competence in vitro and in vivo of bovine IVF blastocyst after 15 years of vitrification Yi Fang, Shenming Zeng, Xiangwei Fu, Baoyu Jia, Shujing Li, Xiaorong An, Yongfu Chen and Shien Zhu	232-238
	A sugar pretreatment as a new approach to the Me2SO- and xeno-free cryopreservation of human mesenchymal stromal cells Yuri A. Petrenko, Olena Y. Rogulska, Vitalii V. Mutsenko and Alexander Y Petrenko	239-246
	Overwintering of the boreal butterfly Colias palaeno in central Europe Pavel Vrba, Matthias Dolek, Oldřich Nedvěd, Helena Zahradníčková, Cristiana Cerrato and Martin Konvička	247-254
	Development of a PVS2 droplet vitrification method for potato cryopreservation Ana Panta, Bart Panis, Cecilia Ynouye, Rony Swennen and William Roca	255-266

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CryoLetters 35 (3), 162-170 (2014)
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RELATIVE IMPORTANCE OF SEED DRYING RATE, DESICCATION TOLERANCE, AND CRYOTOLERANCE FOR THE CONSERVATION OF ARDISIA ELLIPTICA, A. BRUNNESCENS AND A. VIRENS

Xin Yao ¹, Uromi Manage Goodale ¹, Zhilin Li ², Y. Huang ¹,
X. F. Wang ³, F. Y. Cheng ¹, Y. H. Tan ¹, C. F. Xiao ¹,
and Qinying Lan ^1*

¹Key Laboratory of Tropical Forest Ecology and Germplasm Bank, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, 666303 Yunnan, China.
²College of Horticulture and Landscape, Yunnan Agriculture University, Kunming, Yunnan 650201 China 
³Beijing Forestry University, 100083 Beijing, China
^*Corresponding author email:  lqy@xtbg.org.cn

Abstract

BACKGROUND: The pan-tropical genus Ardisia has more than 400 species and is of high horticultural and medicinal value. Due to overexploitation it is important to conserve the germplasm of this genus.  OBJECTIVE: To investigate the feasibility and methods of cryopreservation for long-term seed storage of three Ardisia species: A. elliptica Thunb., A. brunnescens Walker, and A. virens Kurz. METHODS: We tested whether rapid desiccation can increase desiccation tolerance and cryotolerance, and whether the thawing rate can affect cryopreservation success. Seeds were subjected to three desiccation treatments: 1) activated silica gel at 25 ± 2°C, and 4% relative humidity (RH); 2) saturated NaCl solution in closed jars in 25 ± 2°C and 75% RH; and 3) air-drying at room conditions at 27 ± 2°C and RH 60% for different desiccation durations (12h, 24h, 48h, 96h, and 196h). Seeds were then assessed for desiccation tolerance and cryotolerance after rapid thawing (direct immersion in 36°C water bath for 2 min) or slow thawing (at 27°C for 1 h). RESULTS: For all three species, desiccation method and duration significantly affected cryotolerance (P < 0.0001). Fast desiccation did not improve germination compared to slower desiccation (P < 0.01). Whereas A. elliptica germination was unaffected by desiccation duration, drying time significantly (P < 0.0001) affected germination percentage in the other two species especially after 48h. Although slow thawing improved cryotolerance of A. brunnescens seeds (P <0.05), there was no significant effect of thawing rate on A. elliptica. A. virens seed did not survive cryopreservation. CONCLUSIONS: Cryopreservation protocols of Ardisia species may be species-specific and should be established for each species in the genus so that cryopreservation can be used as a successful conservation strategy.

Keywords: Ardisia, conservation, cryopreservation, desiccation tolerance, drying rate, thawing rate.

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CryoLetters 35 (3), 171-179 (2014)
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RELATION BETWEEN FROST TOLERANCE AND POST-CRYOGENIC RECOVERY IN HYPERICUM SPP.

Linda Petijová¹, Katarína Bruňáková¹, Jiří Zámečník²,
Daniela Zubrická¹, Anna Mišianiková¹, Katarína Kimáková¹
and Eva Čellárová¹*

¹Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Mánesova 23, 041 54 Košice, Slovakia.  ²Crop Research Institute, Drnovská 507, 161 06 Prague 6, Czech Republic
*Corresponding author email: eva.cellarova@upjs.sk

Abstract

BACKGROUND: The species of the Hypericum genus are markedly variable in morphological, physiological and biochemical traits. They significantly differ in their area of distribution, which may determine their natural tolerance to environmental conditions, such as temperature extremes. OBJECTIVE: To test the hypothesis that the species growing worldwide in different regions and altitudes would be better able to withstand cryopreservation than the endemics.  METHODS: The frost tolerance of 10 selected Hypericum species was evaluated. A possible stimulatory effect of cold-acclimation and vitrification-associated stressors on the content of hypericins was also investigated  RESULTS: We found that frost tolerance of 10 selected Hypericum species expressed by LT₅₀ ranged between -11°C for the species occurring worldwide and -4°C for sub/tropical frost sensitive taxons which corresponded with their natural habitats.  CONCLUSIONS: Although the mean recoveries for all species cryopreserved with the same vitrification procedure did not exceed 30%, the effect of genetic predisposition to cold tolerance should be considered for optimisation of cryopreservation protocol. Our data neither proved an elicitation effect of cold on hypericin biosynthesis, nor correlation between hypericin content and quantitative characteristics of the hypericin-accumulating black nodules.

Keywords: black nodules, cold acclimation, frost tolerance, hypericin, vitrification

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CryoLetters 35 (3), 180-187 (2014)
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USE OF FM1-43, A MEMBRANE-SPECIFIC FLUORESCENT DYE, TO ESTIMATE PLASMA MEMBRANE INTEGRITY IN THE CRYOPRESERVATION OF GREEN ALGAE

Tomokazu Yamazaki^1,2, Chizuru Hirai¹, Shuhei Ota^1,2,
Kazuyoshi Kuwano³, Shigeyuki Kawano^1,2

¹Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
²JST, CREST, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
³Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki, 852-8521, Japan
*Corresponding author email: yamazaki@ib.k.u.-tokyo.ac.jp

Abstract

BACKGROUND AND OBJECTIVE: Cryopreservation of microorganism cultures is an important technology for their use as biological and genetic resources; however, the procedure is complicated and depends on the species. MATERIALS AND METHODS: We used the two-step freezing method for the cryopreservation of the green alga Parachlorella kessleri.  RESULTS AND CONCLUSION: The optimal cryoprotectant for cryopreservation was 5% dimethyl sulfoxide plus 5% ethylene glycol. This is different from the optimal cryoprotectant for the closely related species Chlorella vulgaris. Efficient cryopreservation of P. kessleri was achieved using methanol, similar to Chlamydomonas reinhardtii. A membrane-specific fluorescent dye, FM1-43, was applied to estimate plasma membrane integrity. We found that the plasma membrane integrity of P. kessleri cells after freeze-thawing was associated with survivability, suggesting that this is a useful index for the optimization of the first step of the two-step freezing method of cryopreservation.

Keywords: FM1-43, cryopreservation, two-step freezing, plasma membrane, green algae, Parachlorella kessleri

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CryoLetters 35 (3), 188-196 (2014)
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CRYOPRESERVATION OF QUINCE (CYDONIA OBLONGA MILL.)

Paul T. Lynch^1*, Ayesha Siddika¹, Aradhana Mehra¹,
Carla Benelli² and Maurizio Lambardi²

¹Biological Sciences Research Group, University of Derby, Derby, UK.
²Istituto per la Valorizzazione del Legno e delle Specie Arboree, (IVALSA), National Research Council, (Firenze) Italy.
*Corresponding author email: P.T.Lynch@derby.ac.uk

Abstract

BACKGROUND: Quince (Cydonia oblonga Mill.) has great potential for utilisation in pharmaceutical and food industries. OBJECTIVE: The study was to develop an efficient cryopreservation approach for quince. METHODS: Factors on the survival and regrowth such as cold acclimation, explant type and recovery media composition were assessed. The effectiveness of the resultant protocols for a number of quince cultivars was determined. RESULTS and CONCLUSION: Quince shoot tips and nodal sections are successfully cryopreserved. Sustained regrowth of quince 'Angers A' was observed after encapsulation-osmoprotection/dehydration, encapsulation-dehydration and PVS2 vitrification. The highest regrowth rate (80%) was obtained from explants excised from cold hardened shoots and cryopreserved using encapsulation-osmoprotection / dehydration and vitrification protocols. The optimised vitrification protocol in combination with shoot cold hardening and a MS recovery medium without activated charcoal and auxin resulted in satisfactory regrowth of shoots from six quince cultivars. The morphology of acclimatised plants derived from cryopreserved shoots was comparable with non-cryopreserved plants.

Keywords: quince, shoot tips, nodal sections, cold hardening, recovery medium

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CryoLetters 35 (3), 197-203 (2014)
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GROWTH MEDIUM ALTERATIONS IMPROVE IN VITRO COLD STORAGE OF PEAR GERMPLASM

Irina Kovalchuk¹, Zhangul Zhumagulovа¹, Timur Turdiev¹
and Barbara M. Reed^2*

¹ Institute of Plant Biology and Biotechnology at National Center of Biotechnology, Timiryazev Str. 45, 050040, Almaty, Republic of Kazakhstan. kovalchuk_i_u@mail.ru
² United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333, USA
^*Corresponding author email: Barbara.Reed@ars.usda.gov

Abstract

BACKGROUND: Development of new fruit cultivars is dependent on genetic resource collections such as those at the Pomological Garden of the Institute of Horticulture and Viticulture near Almaty, Kazakhstan. The pear germplasm collection of the Pomological Garden contains 615 cultivars and three species. In vitro cold storage of the collection would provide additional security to the field collection. OBJECTIVE: This study was designed to improve medium-term in vitro storage of pear germplasm. METHODS: Shoots of seven pear cultivars (Pyrus communis L.) were stored in plastic five-section bags at 4°C and a 10-h photoperiod (7 μmol m^-2 s^-1). Treatments included medium with four carbohydrate sources (3% sucrose, 2% or 3% mannitol, or 2% sucrose + 2% mannitol) with 0.5 mg l^-1 BAP and 0.1 mg l^-1 IBA or without plant growth regulators (PGRs) and at three Murashige and Skoog (MS) nitrogen concentrations (100%, 50% or 25%). RESULTS: Pear shoots remained viable for 9 to 15 months without repropagation on the control MS medium with 3% sucrose without PGRs. There were significant impacts of cultivar and treatment on the duration of cold storage. Shoots of 'Mramornaya' remained viable (rating of ≥ 2) for 27 months with PGRs and 2% sucrose + 2% mannitol compared to 12 months for the PGR + 3% sucrose treatment. 'Talgarskaya Krasaviza' stored for 18 months on 2% sucrose + 2% mannitol while all other treatments lasted only 6 to 9 months. Treatments with 0.5 or 1 mg l^-1 abscisic acid (ABA) with 3% sucrose increased storage duration as did reducing the concentration of nitrogen in the medium to 25% without PGRs and with 3% sucrose.

Keywords: germplasm storage, in vitro cold storage, mannitol, micropropagation, nitrogen, Pyrus

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CryoLetters 35 (3), 204-215 (2014)
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CRYOPRESERVATION OF PASSIFLORA POHLII NODAL SEGMENTS AND ASSESSMENT OF GENETIC STABILITY OF REGENERATED PLANTS

T. S. M. Merhy^1,2,  M.G. Vianna¹, R.O. Garcia¹,
G. Pacheco¹ and E. Mansur^1*

¹Instituto de Biologia Roberto Alcantara Gomes, Núcleo de Biotecnologia Vegetal, Universidade do Estado do Rio de Janeiro, Rua Săo Francisco Xavier 524 PHLC sala 505, Maracană, Rio de Janeiro 20550-013, Brazil.
²Universidade Federal do Rio de Janeiro, Centro de Cięncias da Saúde Pós-Graduaçăo em Biotecnologia Vegetal, Av. Carlos Chagas Filho, 373, Bloco K, 2° andar, sala 032 - Ilha do Fundăo, Cidade Universitária - Rio de Janeiro 21941-590, Brazil.
*Corresponding author email: elisabeth.mansur@gmail.com

Abstract

Passiflora pohlii is a wild species native to Brazil, with a potential agronomic interest due to its tolerance to soil-borne pathogens that cause damage to the passion fruit culture, and could be used in breeding. Because this species occurs in impacted regions, the goal of this study was the development of in vitro conservation strategies, using nodal segments from axenic plants. Encapsulation-vitrification and vitrification techniques were tested for cryopreservation of nodal segments. The highest recovery (65%) was obtained with the vitrification technique using treatment with the PVS3 vitrification solution from 30 to 120 min. Post-rewarming recovery was achieved on MSM medium supplemented with 30.8 μM BAP with incubation in the dark for 30 days before transfer in the presence of light. No differences were detected between control and cryopreserved materials as assayed by RAPD and ISSR.

Keywords: Passiflora, cryopreservation, nodal segments, somaclonal variation.

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CryoLetters 35 (3), 216-225 (2014)
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PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF PLANTS REGENERATED FROM NON-CRYOPRESERVED AND CRYOPRESERVED WILD SOLANUM LYCOPERSICUM MILL. SEEDS

Byron Zevallos¹, Inaudis Cejas², Florent Engelmann³,
Domenico Carputo⁴, Riccardo Aversano⁴,
Maria-Teresa Scarano⁵, Ermis Yanes⁶,
Marcos Martínez-Montero⁶ and José Carlos Lorenzo⁶

¹Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAM), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador, E-mail: bzevallos@espam.edu.ec.
² Faculty of Agronomy, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba.
³ IRD, UMR DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex, 5, France, E-mail: florent.engelmann@ird.fr
⁴ Department of Agricultural Sciences, University of Naples Federico II, Via Universitŕ 100 - 80055 Portici (NA), Italy, E-mail: raversan@unina.it
⁵ Institute of Plant Genetics (Research Division Portici), National Council of Research, Via Universitŕ 133 - 80055, Portici (NA), Italy, E-mail: mariateresa.scarano@igv.cnr.it
⁶ Laboratory for Plant Breeding, Centro de Bioplantas, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba. E-mail: jclorenzo@bioplantas.cu, cubaplantas@gmail.com URL: www.bioplantas.cu.
*Corresponding author email: bzevallos@espam.edu.ec

Abstract

BACKGROUND: Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. OBJECTIVE: In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. METHODS: Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. RESULT: No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. CONCLUSION: Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.

Keywords: tomato; cryostorage; phenotypic variation, microsatellites

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CryoLetters 35 (3), 226-231 (2014)
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NIV VERSUS DROPPING VITRIFICATION IN CRYOPRESERVATION OF HUMAN OVARIAN TISSUE

Zhun Xiao¹, Shang–Wei Li¹, Yao-Yao Zhang¹, Yan Wang¹,
Ling-ling Li², Wei Fan^1*

¹ Reproductive Medical Center of West China 2^nd University Hospital, Sichuan University, Chengdu 610041;
² Department of Obstetrics and Gynecology of Sichuan Provincial Hospital for Women and Children, Chengdu 610031, China.
^*Corresponding author email: xiaozhunok@163.com

Abstract

BACKGROUND: The containers for vitrification of tissues include cryovials, copper grids, Pasteur pipettes, the solid-surface method and etc. Recently the acupuncture needle was used to achieve better result in vitrification of human ovarian tissue. OBJECTIVE: To determine if the needle immersed vitrification method (NIV) is a promising approach to vitrify the human ovarian tissue.  METHODS: Human ovarian biopsies from five patients were vitrified using NIV and Dropping vitrification. After 14 days of in vitro culture, the incidence of apoptotic primordial follicles from fresh and vitrified groups was assessed by TUNEL assay. 17β-estradiol (E₂) and progesterone (P₄) were detected in the media after culturing of vitrified and fresh ovarian tissues. RESULTS: The incidence of apoptotic primordial follicles was significantly higher in the dropping vitrification group than in the NIV group (P<0.05). E₂ and P₄ concentrations were significantly higher in NIV groups than in Dropping vitrification group (P<0.05).  CONCLUSIONS: NIV was an appropriate method to vitrify ovarian tissue by improving the growth potential of frozen-warmed ovarian tissue in vitro culture.

Keywords: human ovarian tissue, vitrification, primordial follicle, apoptosis, steroid hormone.

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CryoLetters 35 (3), 232-238 (2014)
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DEVELOPMENTAL COMPETENCE IN VITRO AND IN VIVO OF BOVINE IVF BLASTOCYST AFTER 15 YEARS OF VITRIFICATION

Yi Fang¹, Shenming Zeng¹, Xiangwei Fu¹, Baoyu Jia¹,
Shujing Li², Xiaorong An³, Yongfu Chen³ and Shien Zhu^1,*

¹Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R. China.
²Bingjing AnBo Embryo Biotech Center, Beijing 100107, P.R. China.
³College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
^*Corresponding author email: zhushien@cau.edu.cn

Abstract

BACKGROUND: It is uncertain whether long-term cryopreservation affects embryonic development. OBJECTIVE: This study was to investigate the effects of long-term cryopreservation on in vitro and in vivo developmental competence of bovine blastocysts. METHODS: The blastocysts were randomly allocated into 3 groups based on the storage time: 0.5-year group, 1-year group and 15-years group. The thawed blastocysts were subjected to in vitro culture or embryo transplantation. RESULT: Significantly lower survival rate (89.20%) and re-expansion rate (70.27%) of blastocysts were obtained from 15-years group compared with those of 0.5-year (97.50% and 87.50%) and 1-year (100.00% and 84.20%) groups (P < 0.05). There were no significant differences in the hatching rate (39.50% to 42.50%) among the three groups and the pregnancy rate between 1-year (35.00%) and 15-years (36.36%) groups. CONCLUSIONS: Although in vitro developmental competence of the 15 years cryopreserved blastocysts was decreased slightly, the pregnancy outcome was not affected.

Keywords: Bovine, IVF, blastocyst, long-term storage, vitrification

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CryoLetters 35 (3), 239-246 (2014)
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A SUGAR PRETREATMENT AS A NEW APPROACH TO THE Me₂SO- AND XENO-FREE CRYOPRESERVATION OF HUMAN MESENCHYMAL STROMAL CELLS

Yuri A. Petrenko*, Olena Y. Rogulska, Vitalii V. Mutsenko
and Alexander Y. Petrenko

Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine.
*Corresponding author e-mail: yu.a.petrenko@gmail.com

Abstract

Experimental and clinical applications of mesenchymal stromal cells (MSCs) require the development of new approaches and improvements of existing methods of cell cryopreservation. Cryoprotective solutions for effective cell preservation usually contain 10% dimethyl sulfoxide (Me₂SO) and fetal bovine serum (FBS) limiting clinical application of MSCs. We have developed a novel approach to the cryopreservation of human MSCs comprising inclusion of sugars into incubation medium for 24 hrs prior to cryopreservation (pretreatment) with their obligatory presence in cryoprotective solution. Such combined application of mannitol, lactose, sucrose, trehalose or raffinose on cultivation and cryopreservation stages resulted in a significant increase of MSCs viability. Optimal concentration of sugars for cell pretreatment was 200 mM, as an additive in cryoprotective solution – 300 mM. Highest cell viability and metabolic activity assessed by Alamar Blue test were achieved with sucrose, trehalose and raffinose. Using these sugars about 50% of pretreated cells after cryopreservation in the absence of Me₂SO and FBS preserved their survival and metabolic activity. During following recultivation cryopreserved MSCs were able to attachment, proliferation and multilineage differentiation towards osteogenic and adipogenic lineages. The data obtained indicate that the approach included pretreatment of cells with sugars combined with their presence in cryoprotective solution is feasible for future regenerative medicine projects.

Keywords: cryopreservation, pretreatment, mesenchymal stromal cells, sugars, viability, differentiation

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CryoLetters 35 (3), 247-254 (2014)
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OVERWINTERING OF THE BOREAL BUTTERFLY COLIAS PALAENO IN CENTRAL EUROPE

Pavel Vrba¹, Matthias Dolek², Oldřich Nedvěd^1,3*,
Helena Zahradníčková³, Cristiana Cerrato⁴ and Martin Konvička^1,3

¹ Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic;
² Büro Geyer und Dolek, Wörthsee, Germany;
³ Institute of Entomology, Biology Centre, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic;
⁴ Department of Life Science and System Biology, University of Turin, Torino, Italy.
*Corresponding author email: nedved@prf.jcu.cz.

Abstract

BACKGROUND: Colias palaeno (Linnaeus, 1761) (Lepidoptera: Pieridae) is a butterfly with boreal distribution with declining populations in peat bogs and subalpine habitats in Central Europe. OBJECTIVE: We investigated the cold tolerance of overwintering caterpillars from one mountain population from Czech Republic (960m a.s.l.) and one alpine population from Italy (2000m a.s.l.). METHODS: We measured supercooling point (SCP), lower lethal temperature (LLT) and content of cryoprotectants.  RESULTS: The caterpillars were freeze-avoiding, with lower LLT close to their very low SCP (–25 to –27°C). The mountain population accumulated high concentrations of glycerol (5% fresh mass) and sugars (trehalose 0.8%, glucose 0.2%), while the Italian alpine population only moderate amounts of glycerol (0.3%) and sugars (trehalose 0.5%, glucose 0.3%) without effect on their cold hardiness. Larvae that overwintered at +5°C had a lower body mass than those overwintering in natural conditions, indicating a metabolic weight loss, but both groups survived equally well. CONCLUSION: We hypothesize that the high concentration of glycerol contributes to the high desiccation tolerance.

Keywords: cold hardiness, cryoprotective polyols, insect conservation, mountain ecology

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CryoLetters 35 (3), 255-266 (2014)
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DEVELOPMENT OF A PVS2 DROPLET VITRIFICATION METHOD FOR POTATO CRYOPRESERVATION

Ana Panta^1*, Bart Panis², Cecilia Ynouye¹,
Rony Swennen^2,3 and William Roca¹

¹ International Potato Center (CIP). Apartado 1558, Lima 12, Peru;
² Bioversity International, Willem de Croylaan 42 – box 2455, 3001 Leuven, Belgium;
³ Department of Biosystems, KU Leuven, Willem de Croylaan 42 – box 2455, 3001 Leuven, Belgium; and IITA, International Institute of Tropical Agriculture, POB 10, Duluti, Arusha, Tanzania
^*Corresponding author email: a.panta@cgiar.org

Abstract

BACKGROUND: CIP maintains the largest in vitro clonal potato collection in the world, comprising 4,013 landraces and 3,353 improved accessions. The in vitro technology is more efficient and secure than conservation in the field, allowing in vitro plantlets to be stored for approximately 2 years without sub-culture. This method however is not ideal for the long-term germplasm conservation because it is labor consuming, costly, and carries risks of losing accessions due to human error, such as contamination and mislabeling during sub-culturing. OBJECTIVE: To improve the potato cryopreservation procedure based on the droplet PVS2 vitrification. METHODS: The improved method is as follows: excision of 1.8-2.5 mm apical shoot tips from 3 weeks old cultures; 15 min exposure to a loading solution and 50 min to PVS2 (at 0°C); ultra-rapid cooling on aluminum foil strips (0.5 x 2 cm) in LN; rewarming (20 min) in 1.2 M sucrose MS liquid medium; post-cryo culture in the dark on potato meristem medium with progressively decreased sucrose levels (daily transfers from 0.3, to 0.2, to 0.1 M and maintained on 0.07 M). This method was compared with those previously applied by IPK (Germany) and CIP potato genebanks. RESULTS: Survival and recovery were higher using the PVS2 droplet method. Cultivars from several species, one frost tolerant (Solanum juzecpzukii, cv. Pińaza) and two drought tolerant (S. tuberosum subsp andigena, cv Ccompis, and Solanum spp, cv Desiree) responded similarly. CONCLUSIONS: The improved method is recommended for the long term conservation of diverse potato germplasm.

Keywords: abiotic stress, Andean potatoes, DMS0, genebank, genetic resources, PVS2; Solanum spp.

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