Abstracts: CryoLetters 32 (4), 2011

CryoLetters is a bimonthly, international journal for low temperature science and technology



Volume 32, No. 4 July/August 2011

ISSN 0143-2044



Gelled droplet vitrification improves recovery of cryopreserved potato germplasm
Dai Hirai




Cold tolerance of field-collected and laboratory reared larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae)
Stefanos S. Andreadis, Zisis Vryzas, Euphemia Papadopoulou-Mourkidou and Matilda Savopoulou-Soultani




A novel means for cryopreservation of germplasm of the recalcitrantseeded species, Ekebergia capensis
Elliosha Hajari, Patricia Berjak, N.W. Pammenter
and M. Paula Watt




Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification
K. K. Sajini, Anitha Karun, C. H. Amarnath
and Florent Engelmann




Developing seed cryobank strategies for Tabebuia heptaphylla (bignoniaceae), a hardwood tree of the brazilian south atlantic forest
Taiza Cristina Higa, Maria Terezinha Silveira Paulilo,
Erica E Benson, Ênio Pedrotti and Ana Maria Viana




Study on permeability of dmso in embryos of red seabream (Pagrus major) by capillary electrophoresis
F. Lin, Q. H. Liu, Z. Z. Xiao, D. Y. Ma , S. H. Xu, Y. S. Xiao and J. Li




Lipid content and cryotolerance of porcine embryos cultured with phenazine ethosulfate
Barbara Gajda, Marek Romek, Izabela Grad,
Ewa Krzysztofowicz, Magdalena Bryła and Zdzisław Smorąg




Cryopreservation of winter-dormant apple buds: I - variation in recovery with cultivar and winter conditions
C.Vogiatzi, B.W.W.Grout, A.Wetten
and T.B.Toldam-Andersen





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CryoLetters 32 (4), 287-296 (2011)
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Gelled droplet vitrification improves recovery of cryopreserved potato germplasm

Dai Hirai

Central Agricultural Experiment Station, Hokkaido Research Organization, 363-2, Minami-takinokawa, Takikawa, Hokkaido, Japan 073-0013
Corresponding author email:


The droplet vitrification method was improved for maneuverability by embedding shoot tips in gelled droplets before osmoprotection. This newly modified cryopreserving method  –gelled droplet vitrification –  was compared with other PVS2-based cryopreservation methods using potato shoot tips. Survival rates of each cryogenic procedure held at 25ºC were about 40% by cryotube-vitrification procedures (vitrification and encapsulation vitrification methods) and about 70% by PVS2-droplet procedures (droplet vitrification and gelled droplet vitrification methods). Much higher cooling rates of PVS2-droplet procedures than cryotube- vitrification procedures increased their survival rates. The gelled droplet vitrification method was applied to shoot tips of 26 potato cultivars and six wild potatoes. After a little modifications of the conditions for preculture, osmoprotection and dehydration, all cultivars and wild potatoes produced high enough survival rates to be of value to genebanks and all surviving shoot tips developed normal shoots within 3 weeks.

Keywords: vitrification, encapsulation vitrification, droplet vitrification, gelled droplet vitrification



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CryoLetters 32 (4), 297-307 (2011)
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Stefanos S. Andreadis1, Zisis Vryzas2, Euphemia Papadopoulou-Mourkidou2 and Matilda Savopoulou-Soultani1*

1Laboratory of Applied Zoology and Parasitology, Department of Plant Protection, Faculty of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
2Laboratory of Agricultural Pharmacology and Ecotoxicology, Department of Agricultural Development, Democritus University of Thrace, 68 200 Orestiada, Greece.
3Pesticide Science Laboratory, Department of Plant Protection, Faculty of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
*Corresponding author  email:


Sesamia nonagrioides Lefébvre (Lepidoptera: Noctuidae) is considered one of the most destructive pests of corn in the Mediterranean region. The purpose of the present study was to investigate some aspects of the cold tolerance of non-diapausing and diapausing laboratory reared larvae of S. nonagrioides, as well as of field-collected larvae, taking into consideration various parameters, such as supercooling ability, mean lethal temperature and accumulation of cryoprotectant substances, in relation to diapause. Our results provide evidence that S. nonagrioides has limited cold tolerance as it displays a low ability of supercooling. This is strongly supported by the fact that mortality of the individuals occurred after extended exposure to subzero temperatures, equivalent or slightly lower to their mean supercooling point. However, lethal temperatures of diapausing larvae were significantly lower in relation to that of non-diapausing larvae, indicating the existence of a direct link between diapause and cold tolerance. Regarding the role of cryoprotectant substances, accumulation of glycerol seems to be closely related to diapause, in contrast to accumulation of trehalose, which is more related to exposure to low temperatures slightly higher than 0oC. Finally, non-diapausing larvae of different instars displayed a similar ability of supercooling and tolerance to low temperatures as well as accumulation of cryoprotectant substances. The ecological significance of our findings on cold tolerance of this species is being discussed with particular reference to the microclimate observed in northern Greece.

Keywords: cold hardiness, diapause, glycerol, lower lethal temperature, Sesamia nonagrioides, supercooling point, trehalose.



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CryoLetters 32 (4), 308-316 (2011)
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Elliosha Hajari, Patricia Berjak*, N.W. Pammenter and M. Paula Watt

School of Biological & Conservation Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa
*Corresponding author e-mail:


Cryopreserved zygotic embryonic axes offer the best means of genetic diversity conservation of recalcitrant-seeded species, but frequently shoots fail to develop following processing for, and after, cryostorage. The present work offers a means to overcome this, by generating adventitious shoots from seedling roots produced after axis cryopreservation. Embryonic axes of Ekebergia capensis were exposed to cryoprotectants, flash dried, and rapidly cooled in nitrogen slush. Cryoprotection was an essential step, with both glycerol and DMSO permitting survival after cryogen exposure, but sucrose alone, or in combination with glycerol, was deleterious. Adventitious shoots were formed from seedling roots developed by axes germinated after cryogen exposure, after being subjected to intermittent flushing with a BAP-containing medium for 24 h in a temporary immersion system and subsequent culture on a semi-solid BAP-containing medium. After excision, a high proportion of the adventitious shoots produced roots in vitro, with most of these rooted plantlets being subsequently successfully acclimated.

Keywords: adventitious shoots; cryoprotection; plantlets; post-cryopreservation; recalcitrant seeds; temporary immersion.



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CryoLetters 32 (4), 317-328 (2011)
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K. K. Sajini1, Anitha Karun1*, C. H. Amarnath2 and Florent Engelmann3,4

1Biotechnology Section, Central Plantation Crops Research Institute, Kasaragod-671124, Kerala, India.
2Statistics Section, Central Plantation Crops Research Institute, Kasaragod-671124, Kerala India.
3Institut de Recherche pour le Développement (IRD), UMR DIAPC, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France.
4Bioversity International, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.
*Corresponding author  email:


The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80% survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25% of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

Keywords: Cocos nucifera L., coconut, zygotic embryo, preculture, vitrification, PVS3, unloading.



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CryoLetters 32 (4), 328-338 (2011)
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Taiza Cristina Higa1, Maria Terezinha Silveira Paulilo1, Erica E Benson2, Ênio Pedrotti3 and Ana Maria Viana1*

1Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, 88049-070, Brazil.
2Erica E. Benson, Damar Research Scientists, Damar, Drum Road, Cuparmuir, Fife, Scotland, UK, KY15 5RJ.
3Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, SC, 88049-070, Brazil.
*Corresponding author email:


The conservation of Tabebuia heptaphylla, an economically significant, endangered tree of the South Atlantic Forest is confined to arboreta. Although its seeds are orthodox, they do not withstand long-term storage in conventional seed banks, motivating the development of  cryopreservation for this species. Seeds within the moisture content (MC) range of 7.5% (0.08 gH2O.gDW-1) to 8.4% (0.09 gH2O.gDW-1) germinated after storage in liquid nitrogen (LN). Storage duration (15 min to 26 weeks) and rewarming regime (slow and rapid) did not significantly influence germination, which ranged between 54-67%. As no additional cryoprotective treatments were required, the protocol is time-, cost- and technically-efficient. Because transport of seeds in LN is problematic for safety, logistic and technical reasons, the feasibility of implementing germplasm transfer using T. heptaphylla seeds recovered from cryobanks was also tested. Viability was not negatively affected in seeds that had been rewarmed, recovered and maintained at room temperature for 2 weeks, allowing safe germplasm transfer in the unfrozen state. The vigor of seedlings from cryopreserved seeds, which was evaluated 90 days after transfer to soil was not influenced by LN storage compared to the controls.

Keywords: Brazilian hardwoods; ex situ conservation; Atlantic Forest; cryobank.



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CryoLetters 32 (4), 339-348 (2011)
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study on permeability of dmso in EMBRYOS OF red seabream (pagrus major) by capillary electrophoresis

F. Lin1,2,3, Q. H. Liu 1,3, Z. Z. Xiao 1, D. Y. Ma 1 , S. H. Xu, Y. S. Xiao and J. Li 1

1Center of Biotechnology R&D, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P.R. China.
2Graduate school of Chinese Academy of Sciences, Beijing 100049, P.R. China.
3 These authors contribute equally to this work.
Corresponding author:
Tel: 86-532-82898718; Fax: 86-532-82898718.


The objectives were to investigate the permeability of DMSO to red seabream (Pagrus major) embryos by capillary electrophoresis and the effects of DMSO concentrations (5%-40%, v/v) and immersion times (10, 30 and 60 min) on hatching rate and morphology. The results suggested the internal DMSO concentrations were positively related with the external concentrations and exposure times, while the hatching rate was negatively related. The hatching rate decreased drastically (<50%) after exposure in 35%, 20% and 15%  DMSO for over 10, 30 and 60 min, respectively. In all groups, when hatching rate was >50%, the internal DMSO concentration was < 2%, which was still insufficient for successful cryopreservation. Morphological changes indicated the chorion was permeable to the cryoprotectant. A sign of dehydration in yolk were observed, for a significant decrease in the maximal yolk sac diameter. However, further research was needed to investigate whether the DMSO permeated into the yolk.

Keywords: Pagrus major, DMSO permeability, capillary electrophoresis, hatching rate, morphology changes



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CryoLetters 32 (4), 349-357 (2011)
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Barbara Gajda*1, Marek Romek2, Izabela Grad1, Ewa Krzysztofowicz2, Magdalena Bryła1 and Zdzisław Smorąg1


1Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, ul. Krakowska 1, 32-083 Balice/Krakow, Poland,
2Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, ul. R. Ingardena 6, 30-060 Kraków, Poland
*Corresponding author email:


The addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on pig embryo development, apoptosis, cytoplasmic lipid content and survival after OPS vitrification. Porcine zygotes were cultured in NCSU-23 medium supplemented with 0 (control) or 0.05 µM PES up to the blastocyst stage and were vitrified using OPS technology. Culture of embryos with PES reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (45.2 and 37.9%, respectively). These results showed that culturing porcine embryos in medium with PES increased the proportion of morula and blastocyst formation and reduced the index of DNA fragmentation and the cytoplasmic lipid content of cultured blastocysts. However, the use of PES during in vitro culture had limited effect on porcine blastocyst survival after vitrification.

Keywords: pig; embryo; culture in vitro; lipid content; phenazine ethosulfate; vitrification



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CryoLetters 32 (4), 358-366 (2011)
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cryopreservation of winter-Dormant apple buds: I - VARIATION IN RECOVERY with cultivar and winter conditions

C.Vogiatzi1, B.W.W.Grout1*, A.Wetten2 and T.B.Toldam-Andersen1

1Department of Agriculture and Ecology, University of Copenhagen, Højbakkegård Allé   13, DK-2630 Taastrup, Denmark
2School of Biological Sciences, University of Reading, Harborne Building, RG6 6AS, United Kingdom
*Corresponding author email:


The widely-adopted protocol for the cryopreservation of winter buds of fruit trees, such as Malus and Pyrus, was developed in a region with a continental climate, that provides relatively hard winters with a consequent effect on adaptive plant hardiness. In this study the protocol was evaluated in a typical maritime climate (eastern Denmark) where milder winters can be expected. The survival over two winters was evaluated, looking at variation between seasons and cultivars together with the progressive reduction in survival due to individual steps in the protocol. The study confirms that under such conditions significant variation in survival can be expected and that an extended period of imposed dehydration at -4oC is critical for bud survival. The occurrence of freezing events during this treatment suggests that cryodehydration may be involved, as well as evaporative water loss. To optimize the protocol for maritime environments, further investigation into the water status of the explants during cryopreservation is proposed.

Keywords: Malus x domestica, cryopreservation, dormant bud, survival, grafting

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