- Email: [email protected]
83. The relativistic permeability method: A complete physical description of osmotic processes
402
Abstracts / Cryobiology 53 (2006) 367–446
(glycerol 50% (w/v) + sucrose 50% in MS medium) for 90– 150 min, cooling primordia in 5 ll droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 min. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4% and 95.4%. This protocol is currently being applied to Korean garlic collections, which comprise over one thousand accessions, for implementation of germplasm cryopreservation. (Conflict of interest: None declared. Source of funding: None declared).
metal cations in the recovery medium. However, in the short-term recovery phase (e.g. 24–38 h), this effect may be tolerated and negated due to the beneficial effects of the chelator in its capacity to reduce free-radical damage to the cell. This is as evidenced by an enhancement in qualitative recovery manifest as a reduction in bleaching and cell necrosis. Intracellular putrescine levels were similarly shown to decrease during cell stress, due to its catabolism into spermine and spermidine, and also its role as a potential free-radical scavenger. It is recommended that desferrioxamine treatment is restricted to the initial stage of post-storage recovery, a point at which free radical damage will be expected to be at its greatest. After that the cultures should be returned to cation-supplemented and desferrioxamine-free medium before the effects of nutrient limitation stress occurs. (Conflict of interest: None declared. Source of funding: Commission of the European Communities (Project Number QLK5-CT-2002-01279).)
doi:10.1016/j.cryobiol.2006.10.081 doi:10.1016/j.cryobiol.2006.10.082 81. Effect of desferrioxamine on olive somatic embryo tissue regrowth after cryopreservation. Susan M. Trigwell a, Ayesha Siddika a, Jason W. Johnston b, Keith Harding c, Erica E. Benson c, Jan M.C. Geuns d, Alan J. Hargreaves e, Philip L.R. Bonner e, Paul T. Lynch a, a Biological Sciences Research Group, School of Science, University of Derby, Derby DE22 1GB, UK; b HortResearch, 120 Mt Albert Road, Private Bag 92, 169 Auckland, New Zealand; c DAMAR, Conservation, Environmental Science and Biotechnology, Cu-parmuir, Fife KY15 5RJ, UK; d Laboratory of Tropical Crop Improvement, K.U. Leuven, B-3001 Leuven, Belgium; e School of Biomedical and Natural Sciences, Nottingham Trent University, Nottingham NG11 8NS, UK The inclusion of the divalent cation chelator desferrioxamine in post-thaw culture medium is proposed to reduce levels of reactive oxygen species generated by Fenton chemistry, therefore potentially enhancing recovery after cryogenic storage. However, the antioxidant effects of the chelator must be balanced with its potential impact on reducing the availability of essential cations such as iron. Olive somatic embryos cryopreserved by controlled-rate cooling and the inclusion of 100 mg/L desferrioxamine in recovery medium, demonstrated qualitative improvements in vigour (e.g., reduced photo oxidation, cell necrosis) whereas, quantitative recovery (as determined by changes in tissue fresh weight) did not increase significantly. Biochemical markers (transglutaminase, putrescine) were tested as determinants for the physiological basis of recovery. Specifically, transglutaminase (TG)-mediated protein crosslinking since TG enzymes catalyse the post-translational modification of proteins by forming intra- and inter-molecular protein crosslinks, or by primary amine incorporation. Tubulin is a recognised substrate for enzymes and stabilisation of the cytoskeleton by TG-mediated intermolecular crosslinking may occur under certain stresses. The possibility that this occurs in cryopreserved plant cells is considered. Olive somatic embryo tissue analysis revealed an accumulation of high molecular weight (crosslinked) tubulin in samples recovered from cryopreservation, with maximal crosslinking occurring after recovery on cationfree/desferrioxamine culture medium. This stabilisation may be a factor in the reduced quantitative recovery (fresh weight gain) in the presence of desferrioxamine, suggesting that the chelator impairs cell growth presumably by reducing the availability of
82. Towards developing an understanding of the response of Eucalyptus tissues to desiccation in vitro. David J. Mycock a, Paula Watt b, a School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, 2050 Johannesburg, South Africa; b School of Biological and Conservation Sciences, University of KwaZulu-Natal, 4000 Durban, South Africa The forestry industry is an important component of the South African economy and is based on the cultivation of selected Eucalyptus and Pinus spp. The industry has adopted a multifaceted approach to the selection, production and conservation of the plant material and modern biotechnological techniques are being applied. Several mass propagatory approaches have been adopted in the production lines for the Eucalypts including in vitro micropropagation. Cryostorage has application in the regulation of the in vitro production lines and also in the conservation of the often highly selected clonal material. The scientific understandings of the effects of cryostorage on the various Eucalyptus in vitro materials are presently being considered. The in vitro material is at high water concentrations and the tissues are also sensitive to desiccation: it is therefore essential to ascertain the correct preparative procedures for cryostorage. The central stress on the tissues in the cryoprocedures is desiccation. The paper will detail some of the ultrastructural and biochemical responses of the in vitro tissues to desiccation and also outline approaches that are being developed to ameliorate and/or overcome the responses. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.083
Cell-free systems 83. The relativistic permeability method: A complete physical description of osmotic processes. Igor I. Katkov, CELLTRONIX, 92126 San Diego, CA, USA The relativistic permeability (RP) method for osmotic modeling was introduced in 1998 [Cryobiology 37 439–442], and developed further in 2000, 2003 and 2005 [Cryobiology 40 64–83; 41 36–37; 44 193–203; 51 79–80]. It allows great simplification and thoroughly analyzes mathematical aspects of the cell osmotic
Abstracts / Cryobiology 53 (2006) 367–446
403
where a, b (both <0), and C1 C4 are simple functions of the initial and final (equilibrium) conditions. From this standpoint, one exponent represents the first (fast) phase while the second exponent corresponds to the second (slow) phase of osmotic reaction. In general, such bi-exponential relationships represent either a monotonic function that approaches to its final (equilibrium) value with p (and q) ! 0, or it can be a bi-phasic function with a transient extremum (maximum or minimum). There is ‘‘mathematical equality’’ between w and x; thus, we can expect similar (though mutually exclusive) mathematical patterns of behavior. On the other hand, we have shown before that for such 2-solute systems there are FOUR possible physical patterns of osmotic reaction [Problem Cryobiol.2 (1999) 3–11]. One is the ‘‘classical’’ case when water behaves in bi-phasic manner, while movement of CPA is in one direction only. For addition of CPA, we observe shrinkage-reswelling and the minimum of water volume, while CPA moves monotonically into the cell during both phases. For dilution of CPA, the situation is symmetrically opposite but still water moves in and out of the cell while CPA moves only outward. We have thoroughly investigated the point of maximum volume excursion before. However, we also predicted in 1999 what could be an opposite situation, in which the solute behaves bi-phasically while water either moves only out or into the cell. During addition, CPA moves into the cell first, exceeds its extracellular value, then starts escaping the cell, thus reaching its transient extremum. In contrast, water only escapes the cell so it shrinks monotonically. We called this effect ‘‘hypersaturation’’. Similarly, during dilution we could observe an intracellular minimum of CPA, after which it begins returning to the cell, while water moves only inward (effect of ‘‘hyperdilution’’). We also postulated in 1999 that there could be a case, in which either water or the permeable solute movement is bi-phasic depending on the critical value of crp_cr, so the cell either behaves in ‘‘classical’’ way with maximum volume excursion, or hypersaturation/ hyperdilution with transient extremum of the solute occurs. Yet another, the fourth case, is a situation when both water and CPA moves out of the cell (‘‘squeeze’’ effect) during dilution, or cells swells during addition of permeable CPA (‘‘stretch’’ effect). Here we show how the mathematical bi-exponential description corresponds to the four possible physical scenarios, thus, concluding development of the RP approach. One- and multi-solute systems can be analyzed similarly. (Conflict of interest: None declared. Source of funding: None declared.)
84. Cooling of thermal beams of water molecules by time varying electric fields: A theoretical calculation. Ramon Risco, Juan Tubio, CryoBioTech: Escuela Superior de Ingenieros, University of Seville, 41092 Seville, Spain The concentration of cryoprotectant needed to vitrify an organ is inversely related to its cooling rate. If the heat is transferred to its surrounding environment by thermal conduction, the dimensions of the organ severely limits this cooling rate. The possibility of transferring the energy to the electromagnetic field could open the possibility of faster cooling. Here we present a quantum mechanical calculation of the cooling of water molecules in the gas state by time-varying gradients of the electric field. When a set of (neutral) dipoles is embedded in a uniform electric field, the net electric force acting on each dipole is zero, even if the field varies with time. However if the electric field is not uniform the gradient exerts a force on each dipole that is proportional to the gradient of the field. Recently, researches have applied the consequences of this idea to cool cesium atoms [J. Maddi, T.P. Dinneen, H. Gould, Slowing and cooling polar molecules in time-varying electric field gradients, Phys. Rev. A 60 (1999) 3882]. The dipolar character of the water molecule makes it in principle possible to apply this approach to a beam of water molecules in the gas state. If a big set of water molecules in the gas state is contained in a recipient, the width of the Maxwell–Boltzmann velocity distribution is a measure of the temperature of the system. When the container is open, all the molecules escape in the form of a beam. After some time a correlation is established between the speed of each molecule and its position in the beam: the faster ones travel in the front and the slower in the rear. Now this velocity-position correlated beam is made to pass between the plates of a discharged plane condenser; when all the molecules are inside the condenser is charged. An electric field acts on each molecule, but because they are electrically neutral, no net force acts on any of them. However, as the water molecules start to leave the condenser, they enter into a region of zero electric field. Crossing the border of the condenser means having an inward border (gradient) force being acting towards the capacitor. If the electric field inside does not change in time, all the molecules feel the same force because the gradient is the same for all of them. However, if we reduce the electric field of the capacitor at the same time as the molecules are crossing the border, the fastest (front) molecules experience a bigger gradient (force) inwards than the (slower) rear ones. This reduces the width of the velocity distribution and therefore the temperature of the beam. Several effects were taken into account to compute the final temperature of a water molecule beam that was initially at 298 K. In a first approximation we ignore the possible rotational energy of each molecule, giving a final temperature of the beam of 55 K after the cooling process. When we take into account their rotational energy through the quantum Stark Effect, but suppose that all the molecules enter the system in the ground rotational level, the resulting temperature is 166 K. Finally, if we consider the fact that the molecules enter the cooling system with a rotational energy distribution, the temperature achieved for the water molecules is 251 K. Of course, the cooling process can be repeated until the desired temperature is achieved. (Conflict of interest: None. Source of funding: Junta de Andalucia (Spain).)
doi:10.1016/j.cryobiol.2006.10.084
doi:10.1016/j.cryobiol.2006.10.085
response. It operates with normalized values of osmotic variables that is, relative to isotonic and with relative membrane permeability
crp Ps =ðLp RTMiso Þ: In this presentation, we show that for a ‘‘standard’’ two-solute system (permeable CPA + impermeable entities) both the movement of the water (w) and permeable solute (x) over time (q) can be described as bi-exponential functions of an intermediate parameter p (w ” dq/dp):
w ¼C1 eap þ C2 ebp ; x ¼C3 eap þ C4 ebp ;
Abstracts / Cryobiology 53 (2006) 367–446
(glycerol 50% (w/v) + sucrose 50% in MS medium) for 90– 150 min, cooling primordia in 5 ll droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 min. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4% and 95.4%. This protocol is currently being applied to Korean garlic collections, which comprise over one thousand accessions, for implementation of germplasm cryopreservation. (Conflict of interest: None declared. Source of funding: None declared).
metal cations in the recovery medium. However, in the short-term recovery phase (e.g. 24–38 h), this effect may be tolerated and negated due to the beneficial effects of the chelator in its capacity to reduce free-radical damage to the cell. This is as evidenced by an enhancement in qualitative recovery manifest as a reduction in bleaching and cell necrosis. Intracellular putrescine levels were similarly shown to decrease during cell stress, due to its catabolism into spermine and spermidine, and also its role as a potential free-radical scavenger. It is recommended that desferrioxamine treatment is restricted to the initial stage of post-storage recovery, a point at which free radical damage will be expected to be at its greatest. After that the cultures should be returned to cation-supplemented and desferrioxamine-free medium before the effects of nutrient limitation stress occurs. (Conflict of interest: None declared. Source of funding: Commission of the European Communities (Project Number QLK5-CT-2002-01279).)
doi:10.1016/j.cryobiol.2006.10.081 doi:10.1016/j.cryobiol.2006.10.082 81. Effect of desferrioxamine on olive somatic embryo tissue regrowth after cryopreservation. Susan M. Trigwell a, Ayesha Siddika a, Jason W. Johnston b, Keith Harding c, Erica E. Benson c, Jan M.C. Geuns d, Alan J. Hargreaves e, Philip L.R. Bonner e, Paul T. Lynch a, a Biological Sciences Research Group, School of Science, University of Derby, Derby DE22 1GB, UK; b HortResearch, 120 Mt Albert Road, Private Bag 92, 169 Auckland, New Zealand; c DAMAR, Conservation, Environmental Science and Biotechnology, Cu-parmuir, Fife KY15 5RJ, UK; d Laboratory of Tropical Crop Improvement, K.U. Leuven, B-3001 Leuven, Belgium; e School of Biomedical and Natural Sciences, Nottingham Trent University, Nottingham NG11 8NS, UK The inclusion of the divalent cation chelator desferrioxamine in post-thaw culture medium is proposed to reduce levels of reactive oxygen species generated by Fenton chemistry, therefore potentially enhancing recovery after cryogenic storage. However, the antioxidant effects of the chelator must be balanced with its potential impact on reducing the availability of essential cations such as iron. Olive somatic embryos cryopreserved by controlled-rate cooling and the inclusion of 100 mg/L desferrioxamine in recovery medium, demonstrated qualitative improvements in vigour (e.g., reduced photo oxidation, cell necrosis) whereas, quantitative recovery (as determined by changes in tissue fresh weight) did not increase significantly. Biochemical markers (transglutaminase, putrescine) were tested as determinants for the physiological basis of recovery. Specifically, transglutaminase (TG)-mediated protein crosslinking since TG enzymes catalyse the post-translational modification of proteins by forming intra- and inter-molecular protein crosslinks, or by primary amine incorporation. Tubulin is a recognised substrate for enzymes and stabilisation of the cytoskeleton by TG-mediated intermolecular crosslinking may occur under certain stresses. The possibility that this occurs in cryopreserved plant cells is considered. Olive somatic embryo tissue analysis revealed an accumulation of high molecular weight (crosslinked) tubulin in samples recovered from cryopreservation, with maximal crosslinking occurring after recovery on cationfree/desferrioxamine culture medium. This stabilisation may be a factor in the reduced quantitative recovery (fresh weight gain) in the presence of desferrioxamine, suggesting that the chelator impairs cell growth presumably by reducing the availability of
82. Towards developing an understanding of the response of Eucalyptus tissues to desiccation in vitro. David J. Mycock a, Paula Watt b, a School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, 2050 Johannesburg, South Africa; b School of Biological and Conservation Sciences, University of KwaZulu-Natal, 4000 Durban, South Africa The forestry industry is an important component of the South African economy and is based on the cultivation of selected Eucalyptus and Pinus spp. The industry has adopted a multifaceted approach to the selection, production and conservation of the plant material and modern biotechnological techniques are being applied. Several mass propagatory approaches have been adopted in the production lines for the Eucalypts including in vitro micropropagation. Cryostorage has application in the regulation of the in vitro production lines and also in the conservation of the often highly selected clonal material. The scientific understandings of the effects of cryostorage on the various Eucalyptus in vitro materials are presently being considered. The in vitro material is at high water concentrations and the tissues are also sensitive to desiccation: it is therefore essential to ascertain the correct preparative procedures for cryostorage. The central stress on the tissues in the cryoprocedures is desiccation. The paper will detail some of the ultrastructural and biochemical responses of the in vitro tissues to desiccation and also outline approaches that are being developed to ameliorate and/or overcome the responses. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.083
Cell-free systems 83. The relativistic permeability method: A complete physical description of osmotic processes. Igor I. Katkov, CELLTRONIX, 92126 San Diego, CA, USA The relativistic permeability (RP) method for osmotic modeling was introduced in 1998 [Cryobiology 37 439–442], and developed further in 2000, 2003 and 2005 [Cryobiology 40 64–83; 41 36–37; 44 193–203; 51 79–80]. It allows great simplification and thoroughly analyzes mathematical aspects of the cell osmotic
Abstracts / Cryobiology 53 (2006) 367–446
403
where a, b (both <0), and C1 C4 are simple functions of the initial and final (equilibrium) conditions. From this standpoint, one exponent represents the first (fast) phase while the second exponent corresponds to the second (slow) phase of osmotic reaction. In general, such bi-exponential relationships represent either a monotonic function that approaches to its final (equilibrium) value with p (and q) ! 0, or it can be a bi-phasic function with a transient extremum (maximum or minimum). There is ‘‘mathematical equality’’ between w and x; thus, we can expect similar (though mutually exclusive) mathematical patterns of behavior. On the other hand, we have shown before that for such 2-solute systems there are FOUR possible physical patterns of osmotic reaction [Problem Cryobiol.2 (1999) 3–11]. One is the ‘‘classical’’ case when water behaves in bi-phasic manner, while movement of CPA is in one direction only. For addition of CPA, we observe shrinkage-reswelling and the minimum of water volume, while CPA moves monotonically into the cell during both phases. For dilution of CPA, the situation is symmetrically opposite but still water moves in and out of the cell while CPA moves only outward. We have thoroughly investigated the point of maximum volume excursion before. However, we also predicted in 1999 what could be an opposite situation, in which the solute behaves bi-phasically while water either moves only out or into the cell. During addition, CPA moves into the cell first, exceeds its extracellular value, then starts escaping the cell, thus reaching its transient extremum. In contrast, water only escapes the cell so it shrinks monotonically. We called this effect ‘‘hypersaturation’’. Similarly, during dilution we could observe an intracellular minimum of CPA, after which it begins returning to the cell, while water moves only inward (effect of ‘‘hyperdilution’’). We also postulated in 1999 that there could be a case, in which either water or the permeable solute movement is bi-phasic depending on the critical value of crp_cr, so the cell either behaves in ‘‘classical’’ way with maximum volume excursion, or hypersaturation/ hyperdilution with transient extremum of the solute occurs. Yet another, the fourth case, is a situation when both water and CPA moves out of the cell (‘‘squeeze’’ effect) during dilution, or cells swells during addition of permeable CPA (‘‘stretch’’ effect). Here we show how the mathematical bi-exponential description corresponds to the four possible physical scenarios, thus, concluding development of the RP approach. One- and multi-solute systems can be analyzed similarly. (Conflict of interest: None declared. Source of funding: None declared.)
84. Cooling of thermal beams of water molecules by time varying electric fields: A theoretical calculation. Ramon Risco, Juan Tubio, CryoBioTech: Escuela Superior de Ingenieros, University of Seville, 41092 Seville, Spain The concentration of cryoprotectant needed to vitrify an organ is inversely related to its cooling rate. If the heat is transferred to its surrounding environment by thermal conduction, the dimensions of the organ severely limits this cooling rate. The possibility of transferring the energy to the electromagnetic field could open the possibility of faster cooling. Here we present a quantum mechanical calculation of the cooling of water molecules in the gas state by time-varying gradients of the electric field. When a set of (neutral) dipoles is embedded in a uniform electric field, the net electric force acting on each dipole is zero, even if the field varies with time. However if the electric field is not uniform the gradient exerts a force on each dipole that is proportional to the gradient of the field. Recently, researches have applied the consequences of this idea to cool cesium atoms [J. Maddi, T.P. Dinneen, H. Gould, Slowing and cooling polar molecules in time-varying electric field gradients, Phys. Rev. A 60 (1999) 3882]. The dipolar character of the water molecule makes it in principle possible to apply this approach to a beam of water molecules in the gas state. If a big set of water molecules in the gas state is contained in a recipient, the width of the Maxwell–Boltzmann velocity distribution is a measure of the temperature of the system. When the container is open, all the molecules escape in the form of a beam. After some time a correlation is established between the speed of each molecule and its position in the beam: the faster ones travel in the front and the slower in the rear. Now this velocity-position correlated beam is made to pass between the plates of a discharged plane condenser; when all the molecules are inside the condenser is charged. An electric field acts on each molecule, but because they are electrically neutral, no net force acts on any of them. However, as the water molecules start to leave the condenser, they enter into a region of zero electric field. Crossing the border of the condenser means having an inward border (gradient) force being acting towards the capacitor. If the electric field inside does not change in time, all the molecules feel the same force because the gradient is the same for all of them. However, if we reduce the electric field of the capacitor at the same time as the molecules are crossing the border, the fastest (front) molecules experience a bigger gradient (force) inwards than the (slower) rear ones. This reduces the width of the velocity distribution and therefore the temperature of the beam. Several effects were taken into account to compute the final temperature of a water molecule beam that was initially at 298 K. In a first approximation we ignore the possible rotational energy of each molecule, giving a final temperature of the beam of 55 K after the cooling process. When we take into account their rotational energy through the quantum Stark Effect, but suppose that all the molecules enter the system in the ground rotational level, the resulting temperature is 166 K. Finally, if we consider the fact that the molecules enter the cooling system with a rotational energy distribution, the temperature achieved for the water molecules is 251 K. Of course, the cooling process can be repeated until the desired temperature is achieved. (Conflict of interest: None. Source of funding: Junta de Andalucia (Spain).)
doi:10.1016/j.cryobiol.2006.10.084
doi:10.1016/j.cryobiol.2006.10.085
response. It operates with normalized values of osmotic variables that is, relative to isotonic and with relative membrane permeability
crp Ps =ðLp RTMiso Þ: In this presentation, we show that for a ‘‘standard’’ two-solute system (permeable CPA + impermeable entities) both the movement of the water (w) and permeable solute (x) over time (q) can be described as bi-exponential functions of an intermediate parameter p (w ” dq/dp):
w ¼C1 eap þ C2 ebp ; x ¼C3 eap þ C4 ebp ;