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Volume 30, No. 3 May/June 2009
ISSN 0143-2044
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CRYO-BANKING OF Prunus mume POLLEN
AND ITS APPLICATION IN CROSS-BREEDING
Ya-Li Zhang, Rui-Dan Chen, Cui-Juan Huang and Yan Liu
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165-170
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IMPROVED
RECOVERY OF CRYOTHERAPY-TREATED SHOOT TIPS FOLLOWING THERMOTHERAPY OF IN VITRO-GROWN STOCK SHOOTS OF RASPBERRY (Rubus idaeus L.)
Qiaochun Wang and Jari P.T. Valkonen
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171-182
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LIMBAL
EXPLANTS FROM CRYOPRESERVED CADAVER HUMAN CORNEAS. IMMUNOFLUORESCENCE AND LIGHT MICROSCOPY OF EPITHELIAL CELLS GROWING IN CULTURE
M. Bratanov, A. Neronov and E. Nikolova
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183-189
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CRYOPRESERVATION
OF CANINE OVARIAN AND TESTICULAR FIBROBLASTS
Il-jeoung Yu, S. P. Leibo, Nucharin Songsasen, Betsy L. Dresser and In-shik Kim
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190-201
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EFFECTS OF
SUCROSE PRECULTURE ON CRYOPRESERVATION BY DROPLET-VITRIFICATION OF STRAWBERRY CULTIVARS AND MORPHOLOGICAL STABILITY OF CRYOPRESERVED PLANTS
I. Pinker, A. Halmagyi and K. Olbricht
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202-211
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SUCROSE
TREATMENT AND EXPLANT WATER CONTENT: CRITICAL FACTORS TO CONSIDER IN DEVELOPMENT OF SUCCESSFUL CRYOPRESERVATION PROTOCOLS FOR SHOOT TIP EXPLANTS OF THE TROPICAL SPECIES Dioscorea rotundata (YAM)
Marian D. Quain, Patricia Berjak, Elizabeth Acheampong and Joseph I. Kioko
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212-223
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CRYOPRESERVATION
OF MARINE DIATOM ALGAE BY ENCAPSULATION-VITRIFICATION
Endong Zhang, Lijia Zhang, Bing Wang, Baoling Yan and Qihua Wang
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224-231
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CRYOPRESERVATION OF Ginkgo biloba CELL CULTURE: EFFECT OF PRETREATMENT WITH SUCROSE AND ABA
Zhi-Wei Lu, Elena V. Popova, Chun-Hua Wu, Eun-Jung Lee, Eun-Joo Hahn and Kee-Yoeup Paek
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232-243
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CryoLettersCryoLetters 30 (3), 165-170 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
CRYO-BANKING OF Prunus mume POLLEN AND ITS APPLICATION IN CROSS-BREEDING
Ya-Li Zhang 1,3, Rui-Dan Chen1,2, Cui-Juan Huang4 and Yan Liu 1,2
1 Landscape Architecture College, Beijing Forestry University,Beijing China.
2 National Floriculture Engineering Research Center,Beijing, China.
3 Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Science, Chinese Academy of Science, Shanghai, China.
4 Huazhong Agricultural University, Wuhan, China.
*Corresponding author: e-mail: yanliu_beijing@yahoo.com.cn
Abstract
The Mei (Prunus mume Sieb. et Zucc.) is one of the most widely used landscape
plants and important germplasm resources in China. The study of pollen cryopreservation and the construction of a pollen cryo-bank have great importance in
Mei research. From 2003 to 2007 we cryopreserved pollen from 51 Mei cultivars. In vitro germination of cryopreserved pollen was not significantly different from that of
fresh pollen, even after four years of storage in liquid nitrogen. Cryopreserved pollen of 19 cultivars was used successfully for intraspecific hybridizations at Wuhan and Beijing in 2005 and 2006.
Keywords: Prunus mume Sieb.et Zucc, cryopreservation, pollen germination, cross-breeding
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CryoLetters 30 (3), 171-182 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
IMPROVED RECOVERY OF CRYOTHERAPY-TREATED SHOOT TIPS FOLLOWING THERMOTHERAPY OF IN VITRO-GROWN STOCK SHOOTS OF RASPBERRY
(Rubus idaeus L.)
Qiaochun Wang1,2* and Jari P.T. Valkonen1
1Department of Applied Biology, P.O. Box 27, FIN-00014 University of Helsinki, Finland
2Key Laboratory of Genetic Improvement of Horticultural Plants of Northwest China,
College of Horticulture, Northwest University of Agriculture and Forestry, Yangling 712100, Shaanxi, P. R. China
*Corresponding author: email: qiaochunwang@nwsuaf.edu.cn
Abstract
Raspberry bushy dwarf virus (RBDV) can be efficiently eradicated from raspberry plants (Rubus idaeus) by a procedure combining thermotherapy and cryotherapy.
However, the bottleneck of this procedure is that, following thermotherapy, cryopreserved shoot tips become chlorotic during regrowth and eventually die after
several subcultures. In addition, survival of heat-treated stock shoots and recovery of cryopreserved shoot tips following thermotherapy are low. The present study focused
towards improving regrowth of cryopreserved raspberry shoot tips following thermotherapy. Results showed that preconditioning stock shoots with salicylic acid
(SA; 0.01-0.1 mM) markedly increased survival of stock shoots after 4 weeks of thermotherapy. Regrowth of cryopreserved shoot tips following thermotherapy was also
significantly enhanced when SA (0.05-0.1 mM) was used for preconditioning stock shoots. Addition of either Fe-ethylenediaminetetracetic acid (Fe-EDTA, 50 mg/l) or Fe-ethylenediaminedi(o)hydroxyphenylacetic acid (Fe-EDDHA, 50 mg/l) to post-culture
medium strongly promoted regrowth and totally prevented chlorosis of shoots regenerated from cryopreserved shoot tips following thermotherapy. Using the
parameters optimized in the present study, about 80% survival of heat-treated stock shoots and about 33% regrowth of cryopreserved shoot tips following thermotherapy
were obtained. Morphology of plants regenerated from cryopreserved shoot tips following thermotherapy was identical to that of control plants, based on observations
of leaf shape and size, internode length and plant height. Optimization of the thermotherapy procedure followed by cryotherapy will facilitate the wider application of
this technique to eliminate viruses which can invade meristems.
Keywords: cryotherapy; Fe-EDTA; raspberry; Rubus idaeus; salicylic acid; shoot tips; thermotherapy.
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CryoLetters 30 (3), 183-189 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
LIMBAL EXPLANTS FROM CRYOPRESERVED CADAVER HUMAN CORNEAS. IMMUNOFLUORESCENCE AND LIGHT MICROSCOPY OF EPITHELIAL CELLS
GROWING IN CULTURE
M. Bratanov, A. Neronov* and E. Nikolova
Institute of Experimental Morphology and Anthropology with Museum, Bulgarian
Academy of Sciences. Acad. G.Bonchev str., bl.25, 1113 Sofia, Bulgaria.
*Corresponding author: email: neronov@hotmail.com
Abstract
The aim of the present study was to determine whether human cadaver corneas, that
were subject to cryopreservation, would be a source of migrating epithelial cells in vitro and what kind of morphological features these cells possess. Limbal explant culture
was used for expanding the epithelial cells. Non-quantitative light microscopical examinations of the cultures within a period of 28 days were carried out. The
phenotype of cultured cells, particularly of the presumed adult stem cell population, was examined by indirect fluorescent immunostaining using antibodies against corneal
stem cell associated markers p63 and vimentin. The effectiveness of the freezing-thawing protocol was confirmed by cultivation of limbal explants taken from non-cryopreserved cadaver corneoscleral rims.
The result clearly showed that limbal tissue, subjected to cryopreservation and long
lasting (up to 12 months) storage in liquid nitrogen, retains the capacity to be source of migrating and proliferating epithelial cells in vitro including the presumed adult stem
cells and transient amplifying cells.
Keywords: cryopreservation, limbal explant culture, light microscopy, immunofluorescence, p63, vimentin.
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CryoLetters 30 (3), 190-201 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
CRYOPRESERVATION OF CANINE OVARIAN AND TESTICULAR FIBROBLASTS
Il-jeoung Yu1*, S. P. Leibo2, Nucharin Songsasen3, Betsy L. Dresser2 and In-shik
Kim1
1College of Veterinary Medicine & Bio-safety Research Institute, Chonbuk National University, Jeonju 561-756, Korea
2Department of Biological Sciences, University of New Orleans; Audubon Center for
Research of Endangered Species, New Orleans, LA 70131, U.S.A.
3Department of Reproductive Sciences, Center for Species Survival, Smithsonian's
Zoological Park, Front Royal, VA 22630, U.S.A.
*Corresponding author: email: iyu@chonbuk.ac.kr
Abstract
To derive a practical procedure to store canine somatic cells, fibroblasts isolated from
testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants,
and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5ºC in a humidified atmosphere of 5% CO2 + 95% air, and then their membrane
integrity was assayed with a double fluorescent stain, Fertilight®. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival
was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70% and functional
survival ranged from 20-40%. With frozen-thawed ovarian cells, the average membrane integrity was 55-75% and the average functional survival was 35-40%. When frozen in
ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P<0.05). These methods should prove useful to preserve cells
collected from canids in the wild.
Keywords: canine, testicular fibroblasts, ovarian fibroblasts, cryopreservation, membrane integrity, functional survival
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CryoLetters 30 (3), 202-211 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
EFFECTS OF SUCROSE PRECULTURE ON CRYOPRESERVATION BY DROPLET-VITRIFICATION OF STRAWBERRY CULTIVARS AND
MORPHOLOGICAL STABILITY OF CRYOPRESERVED PLANTS
I. Pinker1*, A. Halmagyi2 and K. Olbricht3
1Humboldt-University of Berlin, Institute for Horticultural Sciences, A.-Thaer-Weg 1, D-14195 Berlin, Germany.
2Institute of Biological Research, 400015 Cluj-Napoca, Republicii Str. 48, Romania;
3JKI – Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Horticultural and Fruit Crops, Pillnitzer Platz 3a, D-01326 Dresden, Germany.
*Corresponding author: email: ina.pinker@agrar.hu-berlin.de
Abstract
The droplet-vitrification method was applied to shoot tips of micropropagated strawberry plants (Fragaria × ananassa DUCH. cvs. 'Senga Sengana', 'Korona' and
'Aroma'). Shoot tips of 2 - 3 mm in length were precultured in sucrose (0.1, 0.25, 0.5, 0.75, 1.0 M) enriched media for 24 and 48 h. Subsequently, they were transferred into
6 µl droplets of PVS2 vitrification solution for 20 min and plunged into liquid nitrogen. Rapid rewarming was done in liquid medium at room temperature. The highest
recovery rate in all cultivars (60%) was achieved after a preculture with 0.25 M sucrose for 24 h. From the recovered shoot tips not all continued with shoot development and
multiplication. The number of off-types observed in the field was affected by the sucrose concentration. After preculture in 0.1 M sucrose no off-types were observed,
while after preculture in high sucrose concentrations the number of off-types was much higher reaching 20% at the highest sucrose concentration.
Keywords: Cryopreservation, droplet-vitrification, Fragaria × ananassa DUCH.,
germplasm preservation, off-types, recovery, regrowth
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CryoLetters 30 (3), 212-223 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
SUCROSE TREATMENT AND EXPLANT WATER CONTENT: CRITICAL FACTORS TO CONSIDER IN DEVELOPMENT OF SUCCESSFUL CRYOPRESERVATION
PROTOCOLS FOR SHOOT TIP EXPLANTS OF THE TROPICAL SPECIES Dioscorea rotundata (YAM)
Marian D. Quain 1*, Patricia Berjak2, Elizabeth Acheampong3 and Joseph I. Kioko 2
1 CSIR-Crops Research Institute, P.O.BOX 3785 Kumasi, Ghana
2School of Biological and Conservation Sciences, University of KwaZulu-Natal, Durban,
South Africa
3Tissue Culture Laboratory, Department of Botany, University of Ghana, Legon, Accra, Ghana
*Corresponding author: email: md.quain@cropsresearch.org, marianquain@hotmail.com
Abstract
A study was conducted to determine the optimum methods for conditioning explants
to be used in the development of a simple protocol for long-term conservation of the germplasm of Dioscorea rotundata via cryopreservation. Shoot tips from cultures maintained in vitro were exposed to high concentrations of sucrose prior to silica
gel-based dehydration and vitrification solution-based cryopreservation protocols. Explant water contents were determined, and ultrastructural studies were also carried
out. Initially, culturing explants on medium supplemented with 0.3 M sucrose for 3-5 d considerably reduced tissue water content from about 12.2 g g-1 dry mass to between 4.8 and 5.5 g g-1 dry mass before cryoprotection with modified PVS2 (MPVS2) or
silica gel dehydration. Ultrastructural studies indicated that cells had deposits of starch in plastids following sucrose treatments. Survival for D. rotundata shoot tips
treated with MPVS2 vitrification solution, unloaded with 1.0 M sucrose medium and cooled to -70ºC, was 16% for 15 min treatment and 44% for 40 min. After the 40 min
MPVS2 treatment the TTZ test indicated 88% viability retention of explants cooled to -70ºC, and 44% at -196ºC. Plantlet development was obtained for -70ºC-cooled shoot
tips, whereas only callus development occurred from tissues exposed to liquid nitrogen. Explant regeneration was not obtained with air-dehydration techniques. It was
concluded that vitrification-solution based cryopreservation presently offers the best option for conservation of this Dioscorea species.
Keywords: cryopreservation, desiccation, explants, in vitro, vitrification, yam
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CryoLetters 30 (3), 224-231 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
CRYOPRESERVATION OF MARINE DIATOM ALGAE BY ENCAPSULATION-VITRIFICATION
Endong Zhang1, Lijia Zhang1, Bing Wang2, *, Baoling Yan2 and Qihua Wang1
1 Department of Environmental Science, Liaoning Normal University, Dalian 116029, China.
2 Department of Environmental Engineering, Dalian Nationalities University, Dalian
116600,China.
*Corresponding author: email: wangbing-email@163.com
Abstract
In this paper, the cryopreservation of marine diatom algae (Nitzschia closterium f. minutissima Allen and Nelson and Chaetoceros muelleri Lemmermann) using the
encapsulation-vitrification technique was studied. The effect of the composition the vitrification solution, of the concentration of the loading solution, of the duration of
loading and dehydration treatments and of the washing method on viability of algae was investigated. The results showed that PVS2 was suitable for cryopreservation of these diatoms. In the case of N. closterium, the highest viability (73.8%) was obtained
when alginate beads containing the algal cells were loaded with 50% PVS2 for 60 min, dehydrated with 100% PVS2 for 60 min at 0ºC, frozen and rewarmed rapidly and
washed with a 1.2 M sucrose solution for 30 min at 20ºC. In the case of C. muelleri, the highest viability (48.2%) was obtained when alginate beads containing the algal
cells were loaded with 50% PVS2 for 40 min, dehydrated with 100% PVS2 for 60 min at 0ºC, frozen and rewarmed rapidly and washed with a 1.2 M sucrose solution for 30 min at 20ºC.
Key words: Cryopreservation, encapsulation-vitrification, microalgae, Nitzschia closterium f. minutissima Allen and Nelson, Chaetoceros muelleri Lemmermann,
viability
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CryoLetters 30 (3), 232-243 (2009)
© CryoLetters, c/o businessoffice@cryoletters.org
CRYOPRESERVATION OF Ginkgo biloba CELL CULTURE: EFFECT OF PRETREATMENT WITH SUCROSE AND ABA
Zhi-Wei Lu, Elena V. Popova, Chun-Hua Wu, Eun-Jung Lee, Eun-Joo Hahn and Kee-Yoeup Paek*
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Korea.
*Corresponding author e-mail: paekky@cbnu.ac.kr
Abstract
The report describes the impact of preculture with sucrose and sucrose + ABA on
desiccation and cryopreservation tolerance of cell cultures of Ginkgo biloba L., an important landscape and medicinal tree. Callus clumps were incubated on MS medium
supplemented with high sucrose concentrations (up to 24%, w/v), employed alone or with ABA (2-10 mg l-1) for various durations followed by desiccation for 0-240 min and
cryopreservation. The beneficial effect of preculture on regrowth after desiccation without cryopreservation was only observed for the cells with water content of 20% FW
and was not influenced by presence of ABA. However, preculture of calli in presence of ABA resulted in a lower desiccation rate as compared with untreated controls and calli
pretreated with sucrose alone. In calli precultured with sucrose alone, post-thaw regrowth was occasional regardless of the sugar concentration in the medium, while
pretreatment of calli with ABA and sucrose ensured stable regrowth after cryopreservation. The highest post-thaw regrowth of 54% was achieved for calli
precultured on medium supplemented with 10% (w/v) sucrose and 2 mg l-1 ABA for 21 days followed by desiccation for 150 min. The different effects of preculture treatments
on post-thaw regrowth were associated with significant changes in content and in composition of endogenous soluble sugars in calli. Sucrose and glucose accumulated
preferentially in ABA-precultured calli, while the fructose content was higher in calli precultured in absence of ABA. The possible role of preculture on desiccation and cryopreservation tolerance of G. biloba cell cultures is discussed.
Keywords: Ginkgo biloba, callus, cryopreservation, abscisic acid, sucrose preculture, soluble sugar content
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