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147. The gelled droplet vitrification method; an easy and efficient cryopreservation for shoot tips of cultivated and wild potato cultivars
Abstracts / Cryobiology 59 (2009) 370–418 oocyte, a cell volume measurement was taken in PBS as a control before exposure to anisotonic solutions; then, the oocyte was transferred to specified anisosmotic solutions and allowed to equilibrate for 20 min at 24 ± 1 °C before volume measurement. A total of 72 immature and 72 mature oocytes were individually analyzed and averaged across 3 replicates. The results indicated that when exposed to hyperosmotonic or hypoosomotic solutions, both GV and MII bovine oocytes exhibited a ‘‘classic” osmometric response by shrinking or swelling to a constant volume, relative to their isotonic volume. Cell volumes relative to the volume under isotonic conditions were plotted against the reciprocal of osmolality, which was normalized to the isotonic value (Boyle-van’t Hoff plot). Good fits were obtained (R2 > 90%). The osmotically inactive volume (Vb) of MII bovine oocytes was determined as 26.08%, while GV bovine oocytes had a much smaller Vb, i.e., 16.07% of the isotonic cell volume. (Conflicts of interest: None declared. Source of funding: Science Foundation Ireland, the Shanghai Rising-Star Program (07QA14042) of P. R. C, NSFC (50776060), NCET-070559, Program for Eastern Scholar.) doi:10.1016/j.cryobiol.2009.10.158
145. Intracellular ice formation in non-ideal solutions: A model for freezing of cells. *Gang Zhao a,b, Hiroshi Takamatsu a, a Department of Mechanical Engineering, Kyushu University, Fukuoka 819-0395, Japan, b Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, Anhui, PR China Intracellular ice formation (IIF) is one of the most crucial events during the freezing of cells. Since it often causes fatal injury to cells, prediction of IIF is critical in estimating cell survival during freezing and thus in determining the best protocol by a simulation of the freezing process. Modifying the proposed models [1–3], the authors have developed a model that takes into account the biophysical events during freezing, i.e. volume reduction of cells resulting in a change in the supercooling of protoplasm, and also the nucleation and growth of ice crystals [4]. However, since the model assumed ideal dilute solutions, it may not be valid for solutions with higher concentration of CPAs. The objective of this study was to avoid the assumption of ideal solutions as used in our previous model [4] and to expand the conditions that are applicable to prediction. The description of phase equilibrium for NaCl-CPA-water ternary systems proposed by Fahy [5], with their experimental data, was used instead of the assumption of ideal dilute solutions. The nucleation of ice and its growth, coupled with water transport across the cell membrane, were incorporated in the model. The volume of ice formed in the cells was also taken into account. The simulation was conducted for mouse oocyte suspended in NaCl solutions with 2 or 8 M glycerol using the parameters given by Karlsson et al. [3]. The results were compared with our previous model that assumed ideal solutions. At 2 M glycerol, the difference in the results between the two models was negligible at cooling rates higher than 5 °C/min. However, at cooling rates lower than 2.4 °C/min more ice was formed in the cell in the present model; the volume of intracellular ice at 1.2 °C/min in the present model was 27% of the cell volume, which was five times larger than that predicted with the ideal-solution assumption. In contrast at 8 M glycerol solution, the difference between two models was small irrespective of cooling rates because the reduction of cell volume was much smaller than that in the 2 M glycerol solution. The amount of ice formed in the cell was also very much smaller than that in the 2 M glycerol solution; the volume of intracellular ice relative to the cell volume at 3.6 °C/min was 8.2 10 8 in our previous model and 2.9 10 6 in the present model. Even though the absolute value was small, the latter was 35 times larger than the former. Since the threshold for vitrification has been considered to be a volume fraction of intracellular ice of 1 10 6, the difference between the two models may be significant, particularly for estimating the critical cooling rate for intracellular vitrification. Thus the model using non-ideal assumptions is important in the examination of protocols for cryopreservation. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 20 08060), and the National Natural Science Foundation of China (No. 50506029).)
References [1] [2] [3] [4] [5]
Mazur P. J. Gen. Physiol. 1963;47:347–69. Fahy GM. Cryobiology 1981;18:473–82. Karlsson JOM, Cravalho EG, Toner M. J. Appl. Phys. 1994;75:4442–5. Zhao G, Luo DW, Gao DY. AIChE J. 2006;52:2596–606. Fahy GM. Biophys. J. 1980;32:837–50.
411
tec e, Stéphane Dussert a, Laurence Feugey h, Yann Froelicher i, Lydie Fouilhaux c, Franciane Gamiette j, Agnès Grapin k, Michel Grisoni l, Philippe Guérif h, Arnaud Guyader h, Alain Label e, François Luro g, Bernard Moulin e, Martine Muller e, André Peyrière d, Yvon Prigent e, Michel Renard e, Michel Roux-Cuvelier k, Danièle Roques m, Suzia Rubens j, Jocelyne Sapotille l, Catherine Souchet e, David Teyssedre l, a IRD Montpellier, France, b Bioversity International, c INRA Bordeaux, d INRA Montpellier, e INRA Ploudaniel, f IRD la Réunion, g INRA Corse, h INRA Angers, i CIRAD Corse, j INRA Guadeloupe, k INHP Angers, l CIRAD LaRéunion, m CIRAD Guadeloupe In France, cryopreservation is currently used for a limited number of forest tree species, but has been applied yet very little to horticultural or other agricultural species. The CRYOVEG project aims at (1) developing or optimizing cryopreservation techniques in a range of selected species; and (2) establishing a national scientific and technical network of plant biological resource centers (CRBs) using cryopreservation. The project has a network organization, with IRD/INRA Montpellier as the cryopreservation expertise centre and partners in continental France and in overseas departments in charge of genetic resource conservation for various species: INRA Petit Bourg, Guadeloupe (yam); INRA San Giuliano, Corsica (Citrus); INRA Bordeaux (Prunus); INRA Angers (apple and pear); INRA Montpellier (grapevine); INRA Ploudaniel (potato, Brassica); IRD La Réunion (coffee); CIRAD Roujol, Guadeloupe (sugarcane); and CIRAD La Réunion (vanilla, garlic). Deliverables will vary depending on the state-of-the-art of cryopreservation for each species: for potato, yam, sugarcane, garlic, Citrus, Brassica and coffee, optimized protocols will be tested on a range of representative accessions. For grapevine, Prunus, apple, pear and vanilla, operational protocols will be developed. The group will work as a think-tank on the integration of cryopreservation in the national plant genetic resource conservation strategy and an action plan for its implementation will be collectively designed. The project will work towards establishing a French research network on plant genetic resource cryopreservation, open to all partners interested in cryopreservation. The group will prepare research and development projects whose results will be disseminated through European plant genetic resource conservation networks e.g. ECP/ GR). Finally, network members will train French and foreign (from North and South countries) researchers and technicians in cryopreservation techniques. (Conflicts of interest: None declared. Source of funding: This project is supported by a French competitive grant (Call for Projects 2008 ‘‘Biological Resource Centers”) of the Consultative Committee for Biological Resources/Infrastructures in the Biology, Health and Agronomy sectors (CCRB/IBiSA).) doi:10.1016/j.cryobiol.2009.10.160
147. The gelled droplet vitrification method; an easy and efficient cryopreservation for shoot tips of cultivated and wild potato cultivars. *Dai Hirai, Hokkaido Central Agricultural Experiment Station, Naganuma, Hokkaido 069-1395, Japan Shoot tips excised from nodal segments of potato (Solanum tuberosum L. cv Danshakuimo) were successfully cryopreserved by a gelled droplet vitrification method. Ten to 15 precultured shoot tips (1 to 1.5 mm in length) were embedded in a droplet of 2% Na–Alginate solution (15 ll) immobilised on a strip of aluminum foil and gelled with 0.1 M CaCl2 solution. The aluminum foil was transferred to a 2 ml-cryotube and the immobile shoot tips were osmoprotected with MS liquid medium supplemented with 2 M glycerol plus 1.2 M sucrose for 45 min and then dehydrated with PVS2 solution for 60 min at 25 °C. The strip of aluminum foil was directly plunged into liquid nitrogen. Cryopreserved shoot tips were rewarmed and diluted with 1.2 M sucrose in MS liquid medium and then incubated in recovery medium for 3 weeks. The average rate of survival amounted to 78.1 ± 2.9%, the same as that with the droplet vitrification method (69.6 ± 7.6%) and significantly higher than those obtained with the vitrification method (40.8 ± 18.3%), the encapsulation vitrification method (44.1 ± 4.9%) and the droplet method (0%), under their optimal conditions at 25 °C. Besides the high survival rate, shoot tips in gelled droplets on aluminum foil were easier to manipulate than in the droplet vitrification method while exchanging solutions and transfering the shoot tips into the recovery medium after dilution. The gelled droplet vitrification method was applied to the shoot tips of 24 cultivated potato varieties and 5 wild potatoes. With slight modification of the preculture period, the sucrose concentration in the osmoprotectants and the duration of osmoprotection and dehydration with PVS2 solution, over 60% of shoot tips of all cryopreserved cultivated potato varieties and wild potatoes developed into normal shoots within 4 weeks. (Conflicts of interest: None declared. Source of funding: A grant from the NIAS (National Institute of Agricultural Science) Genebank.) doi:10.1016/j.cryobiol.2009.10.161
doi:10.1016/j.cryobiol.2009.10.159
146. Cryopreservation of French plant genetic resource collections (CRYOVEG). *Florent Engelmann a,b, Emilie Balsemin c, Teresa Barreneche c, Philippe Chatelet d, Jean-Eric Chauvin e, Emmanuel Couturon f, Franck Curk g, Marie-Ange Dantec e, Jean-Paul Dan-
148. Cryopreservation of shoot apices of cranberry and highbush blueberry in-vitro cultures. *Daisuke Kami a,b, Takashi Kikuchi b, Keita Sugiyama b, Takashi Suzuki a, a Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan, b National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555, Japan
145. Intracellular ice formation in non-ideal solutions: A model for freezing of cells. *Gang Zhao a,b, Hiroshi Takamatsu a, a Department of Mechanical Engineering, Kyushu University, Fukuoka 819-0395, Japan, b Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, Anhui, PR China Intracellular ice formation (IIF) is one of the most crucial events during the freezing of cells. Since it often causes fatal injury to cells, prediction of IIF is critical in estimating cell survival during freezing and thus in determining the best protocol by a simulation of the freezing process. Modifying the proposed models [1–3], the authors have developed a model that takes into account the biophysical events during freezing, i.e. volume reduction of cells resulting in a change in the supercooling of protoplasm, and also the nucleation and growth of ice crystals [4]. However, since the model assumed ideal dilute solutions, it may not be valid for solutions with higher concentration of CPAs. The objective of this study was to avoid the assumption of ideal solutions as used in our previous model [4] and to expand the conditions that are applicable to prediction. The description of phase equilibrium for NaCl-CPA-water ternary systems proposed by Fahy [5], with their experimental data, was used instead of the assumption of ideal dilute solutions. The nucleation of ice and its growth, coupled with water transport across the cell membrane, were incorporated in the model. The volume of ice formed in the cells was also taken into account. The simulation was conducted for mouse oocyte suspended in NaCl solutions with 2 or 8 M glycerol using the parameters given by Karlsson et al. [3]. The results were compared with our previous model that assumed ideal solutions. At 2 M glycerol, the difference in the results between the two models was negligible at cooling rates higher than 5 °C/min. However, at cooling rates lower than 2.4 °C/min more ice was formed in the cell in the present model; the volume of intracellular ice at 1.2 °C/min in the present model was 27% of the cell volume, which was five times larger than that predicted with the ideal-solution assumption. In contrast at 8 M glycerol solution, the difference between two models was small irrespective of cooling rates because the reduction of cell volume was much smaller than that in the 2 M glycerol solution. The amount of ice formed in the cell was also very much smaller than that in the 2 M glycerol solution; the volume of intracellular ice relative to the cell volume at 3.6 °C/min was 8.2 10 8 in our previous model and 2.9 10 6 in the present model. Even though the absolute value was small, the latter was 35 times larger than the former. Since the threshold for vitrification has been considered to be a volume fraction of intracellular ice of 1 10 6, the difference between the two models may be significant, particularly for estimating the critical cooling rate for intracellular vitrification. Thus the model using non-ideal assumptions is important in the examination of protocols for cryopreservation. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 20 08060), and the National Natural Science Foundation of China (No. 50506029).)
References [1] [2] [3] [4] [5]
Mazur P. J. Gen. Physiol. 1963;47:347–69. Fahy GM. Cryobiology 1981;18:473–82. Karlsson JOM, Cravalho EG, Toner M. J. Appl. Phys. 1994;75:4442–5. Zhao G, Luo DW, Gao DY. AIChE J. 2006;52:2596–606. Fahy GM. Biophys. J. 1980;32:837–50.
411
tec e, Stéphane Dussert a, Laurence Feugey h, Yann Froelicher i, Lydie Fouilhaux c, Franciane Gamiette j, Agnès Grapin k, Michel Grisoni l, Philippe Guérif h, Arnaud Guyader h, Alain Label e, François Luro g, Bernard Moulin e, Martine Muller e, André Peyrière d, Yvon Prigent e, Michel Renard e, Michel Roux-Cuvelier k, Danièle Roques m, Suzia Rubens j, Jocelyne Sapotille l, Catherine Souchet e, David Teyssedre l, a IRD Montpellier, France, b Bioversity International, c INRA Bordeaux, d INRA Montpellier, e INRA Ploudaniel, f IRD la Réunion, g INRA Corse, h INRA Angers, i CIRAD Corse, j INRA Guadeloupe, k INHP Angers, l CIRAD LaRéunion, m CIRAD Guadeloupe In France, cryopreservation is currently used for a limited number of forest tree species, but has been applied yet very little to horticultural or other agricultural species. The CRYOVEG project aims at (1) developing or optimizing cryopreservation techniques in a range of selected species; and (2) establishing a national scientific and technical network of plant biological resource centers (CRBs) using cryopreservation. The project has a network organization, with IRD/INRA Montpellier as the cryopreservation expertise centre and partners in continental France and in overseas departments in charge of genetic resource conservation for various species: INRA Petit Bourg, Guadeloupe (yam); INRA San Giuliano, Corsica (Citrus); INRA Bordeaux (Prunus); INRA Angers (apple and pear); INRA Montpellier (grapevine); INRA Ploudaniel (potato, Brassica); IRD La Réunion (coffee); CIRAD Roujol, Guadeloupe (sugarcane); and CIRAD La Réunion (vanilla, garlic). Deliverables will vary depending on the state-of-the-art of cryopreservation for each species: for potato, yam, sugarcane, garlic, Citrus, Brassica and coffee, optimized protocols will be tested on a range of representative accessions. For grapevine, Prunus, apple, pear and vanilla, operational protocols will be developed. The group will work as a think-tank on the integration of cryopreservation in the national plant genetic resource conservation strategy and an action plan for its implementation will be collectively designed. The project will work towards establishing a French research network on plant genetic resource cryopreservation, open to all partners interested in cryopreservation. The group will prepare research and development projects whose results will be disseminated through European plant genetic resource conservation networks e.g. ECP/ GR). Finally, network members will train French and foreign (from North and South countries) researchers and technicians in cryopreservation techniques. (Conflicts of interest: None declared. Source of funding: This project is supported by a French competitive grant (Call for Projects 2008 ‘‘Biological Resource Centers”) of the Consultative Committee for Biological Resources/Infrastructures in the Biology, Health and Agronomy sectors (CCRB/IBiSA).) doi:10.1016/j.cryobiol.2009.10.160
147. The gelled droplet vitrification method; an easy and efficient cryopreservation for shoot tips of cultivated and wild potato cultivars. *Dai Hirai, Hokkaido Central Agricultural Experiment Station, Naganuma, Hokkaido 069-1395, Japan Shoot tips excised from nodal segments of potato (Solanum tuberosum L. cv Danshakuimo) were successfully cryopreserved by a gelled droplet vitrification method. Ten to 15 precultured shoot tips (1 to 1.5 mm in length) were embedded in a droplet of 2% Na–Alginate solution (15 ll) immobilised on a strip of aluminum foil and gelled with 0.1 M CaCl2 solution. The aluminum foil was transferred to a 2 ml-cryotube and the immobile shoot tips were osmoprotected with MS liquid medium supplemented with 2 M glycerol plus 1.2 M sucrose for 45 min and then dehydrated with PVS2 solution for 60 min at 25 °C. The strip of aluminum foil was directly plunged into liquid nitrogen. Cryopreserved shoot tips were rewarmed and diluted with 1.2 M sucrose in MS liquid medium and then incubated in recovery medium for 3 weeks. The average rate of survival amounted to 78.1 ± 2.9%, the same as that with the droplet vitrification method (69.6 ± 7.6%) and significantly higher than those obtained with the vitrification method (40.8 ± 18.3%), the encapsulation vitrification method (44.1 ± 4.9%) and the droplet method (0%), under their optimal conditions at 25 °C. Besides the high survival rate, shoot tips in gelled droplets on aluminum foil were easier to manipulate than in the droplet vitrification method while exchanging solutions and transfering the shoot tips into the recovery medium after dilution. The gelled droplet vitrification method was applied to the shoot tips of 24 cultivated potato varieties and 5 wild potatoes. With slight modification of the preculture period, the sucrose concentration in the osmoprotectants and the duration of osmoprotection and dehydration with PVS2 solution, over 60% of shoot tips of all cryopreserved cultivated potato varieties and wild potatoes developed into normal shoots within 4 weeks. (Conflicts of interest: None declared. Source of funding: A grant from the NIAS (National Institute of Agricultural Science) Genebank.) doi:10.1016/j.cryobiol.2009.10.161
doi:10.1016/j.cryobiol.2009.10.159
146. Cryopreservation of French plant genetic resource collections (CRYOVEG). *Florent Engelmann a,b, Emilie Balsemin c, Teresa Barreneche c, Philippe Chatelet d, Jean-Eric Chauvin e, Emmanuel Couturon f, Franck Curk g, Marie-Ange Dantec e, Jean-Paul Dan-
148. Cryopreservation of shoot apices of cranberry and highbush blueberry in-vitro cultures. *Daisuke Kami a,b, Takashi Kikuchi b, Keita Sugiyama b, Takashi Suzuki a, a Research Faculty and Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan, b National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555, Japan