[show abstract][hide abstract] ABSTRACT: Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.
[show abstract][hide abstract] ABSTRACT: Imbibing sunflower (Helianthus annuus L., cv. Briosol) seeds at water potentials between -2 MPa and -5 MPa leads to faster (priming) or slower (accelerated ageing) germination depending on the temperature and duration of treatment. Mobilization of food reserves may be associated with the changes in seed vigor. To study this, morphological, biochemical and phase properties of lipid, the major food reserve in sunflower, were compared in freshly harvested (i.e., control), primed and aged sunflower cotyledons using electron microscopy, biochemical analyses and differential scanning calorimetry, respectively. Lipid bodies became smaller and more dispersed throughout the cytoplasm during priming and ageing. Despite ultrastructural changes, there were few measured changes in biochemistry of the neutral lipid component; lipid content, proportion of saturated and unsaturated fatty acids and level of free fatty acids were unchanged in primed and slightly aged seeds, with only severely aged seeds showing a net decrease in polyunsaturated fatty acids and an increase in free fatty acids. Subtle changes in the calorimetric behavior of lipids within sunflower cotyledons were observed. Sunflower lipids exhibited polymorphic crystalline and amorphous solid phases when cooled to <-100 degrees C, but priming decreased the rate of crystallization in vivo and ageing increased the rate of crystallization, but decreased percentage crystallinity. The observed changes in thermal behavior in vivo are consistent with losses and gains, respectively, of interacting non-lipid moieties in the triacylglycerol matrix.
[show abstract][hide abstract] ABSTRACT: Persistence of anhydrous organisms in nature may depend on how long they remain viable in dry environments. Longevity is determined by interactions of humidity, temperature, and unknown cellular factors that affect the propensity for damaging reactions. Here we describe our research to elucidate those cellular factors and to ultimately predict how long a population can survive under extreme conditions. Loss of viability typically follows a sigmoidal pattern, where a period of small changes precedes a cataclysmic decline. The time for viability to decrease to 50% (P50) varied among seed species and among 10 phylogenetically diverse organisms. When stored at elevated temperatures of 35°C and 32% relative humidity (RH), P50 ranged from about a week for spores of Serratia marcescens to several years for fronds of Selaginella lepidophylla. Most of the species studied survived longest at low humidity (10-20% RH), but suffered under complete dryness. Temperature dependencies of aging kinetics appeared similar among diverse organisms despite the disparate longevities. The effect of temperature on seed aging rates was consistent with the temperature dependency of molecular mobility of aqueous glasses, with both showing a reduction by several orders of magnitude when seeds were cooled from 60°C to 0°C. Longevity is an inherited trait in seeds, but its complex expression among widely divergent taxa suggests that it developed through multiple pathways.
Integrative and Comparative Biology 11/2005; 45(5):751-8. · 3.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Seed collections in gene banks are useful for the preservation of wild germplasm, providing inexpensive insurance for species that survive in conventional cold storage (–18 C). Seeds that cannot survive these conditions must be pretreated with cryoprotectants and stored at liquid nitrogen temperatures, which presents unique technical and methodological challenges. Implicit in this approach is the assumption that these added manipulations do not change the genetic diversity of the preserved collections. We used polymorphic microsatellite markers for an endangered aquatic grass, Texas wild rice (Zizania texana), to conduct a preliminary evaluation of the effects of cryogenic preservation of mature embryos on genetic diversity. Using several statistical approaches, we show that allele frequencies did not change in collections of seeds that underwent cryopreservation (cryoprotected) compared to those samples that was not exposed to cryopreservation (control). The retention of the allelic diversity at the five loci examined suggests that there were no significant changes in genetic diversity due to treatments and that these protocols may be appropriate for ex situ conservation of genetically diverse wild germplasm.
[show abstract][hide abstract] ABSTRACT: Though cryogenic storage is presumed to provide nearly infinite longevity to cells, the actual shelf life achieved under ultra-cold temperatures has not been addressed theoretically or empirically. Here, we report measurable changes in germination of dried seeds stored under liquid nitrogen conditions for >10 years. There was considerable variability in the extent of deterioration among species and accessions within a species. Aging time courses for lettuce seeds stored at temperatures between 50 and -196 degrees C were fit to a form of the Avrami equation to determine rate coefficients and predict half-life of accessions. A reduction in the temperature dependency on aging rate, determined as a break in the Arrhenius plot, occurred at about -15 degrees C, and this resulted in faster deterioration than anticipated from extrapolation of kinetics measured at higher temperatures. The break in Arrhenius behavior occurred at temperatures in between the glass transition temperature (28 degrees C) and the Kauzmann temperature (-42 degrees C) and also coincided with a major triacylglycerol phase change (-40 to -7 degrees C). In spite of the faster than anticipated deterioration, cryogenic storage clearly prolonged shelf life of lettuce seeds with half-lives projected as approximately 500 and approximately 3400 years for fresh lettuce seeds stored in the vapor and liquid phases of liquid nitrogen, respectively. The benefit of low temperature storage (-18 or -135 degrees C) on seed longevity was progressively lost if seeds were first stored at 5 degrees C. Collectively, these results demonstrate that lowering storage temperature progressively increases longevity of seeds. However, cryogenic temperatures were not sufficient to stop deterioration, especially if initial stages of aging were allowed to progress at higher storage temperatures. This work contributes to reliable assessments of the potential benefit and cost of different genebanking strategies.
[show abstract][hide abstract] ABSTRACT: The present study investigated the rate of temperature change within axes of Poncirus trifoliata during cooling and warming by various methods. Cooling rates ranged between 0.17 and 170 degree (C per second, and warming rates of 1.25 and 600 degree C per second were measured when axes were warmed at room temperature or in water at 40 degree C, respectively. Partial drying increased the cooling rate within axes in direct contact with the cryogen, but did not affect the cooling or warming rates within axes enclosed in a double layer of lightweight aluminium foil. The procedures described illustrate the orders of magnitude that separate extremes of the range of cooling or warming rates attained using methods commonly employed in cryopreservation studies. Quantifying these rates allows the relationship between cooling rate, water content and survival of hydrated embryonic axes to be explored.
[show abstract][hide abstract] ABSTRACT: Seed collections in gene banks are useful for the preservation of wild germplasm, providing inexpensive
insurance for species that survive in conventional cold storage ()18 �C). Seeds that cannot survive these
conditions must be pretreated with cryoprotectants and stored at liquid nitrogen temperatures, which
presents unique technical and methodological challenges. Implicit in this approach is the assumption that
these added manipulations do not change the genetic diversity of the preserved collections. We used
polymorphic microsatellite markers for an endangered aquatic grass, Texas wild rice (Zizania texana), to
conduct a preliminary evaluation of the effects of cryogenic preservation of mature embryos on genetic
diversity. Using several statistical approaches, we show that allele frequencies did not change in collections of seeds that underwent cryopreservation (cryoprotected) compared to those samples that was not exposed to cryopreservation (control). The retention of the allelic diversity at the five loci examined suggests that there were no significant changes in genetic diversity due to treatments and that these protocols may be appropriate for ex situ conservation of genetically diverse wild germplasm.
[show abstract][hide abstract] ABSTRACT: Many species within the genus Cuphea (Lythraceae) produce seed with high levels of medium-chain fatty acids. Seeds of some Cuphea species lose viability when placed into storage at -18 degrees C. These species tolerate significant drying to 0.05 g/g and may, therefore, be intermediate in their storage characteristics. The thermal properties of seed lipids were observed using differential scanning calorimetry. Species with peak lipid melting temperatures >/=27 degrees C were found to be sensitive to -18 degrees C exposure while those with melting temperatures <27 degrees C were able to tolerate low-temperature exposure. This relationship was determined by the triacylglycerol composition of the individual species. Sensitive species have high concentrations of lauric acid (C(12)) and/or myristic acid (C(14)). Species with high concentrations of capric (C(8)) or caprylic acid (C(10)) or with high concentrations of unsaturated fatty acids tolerate low temperature exposure. Potential damage caused by low temperature exposure can be avoided by exposing seeds to a brief heat pulse of 45 degrees C to melt solidified lipids prior to imbibition. The relationship between the behavior of triacylglycerols in vivo, seed storage behavior and sensitivity to imbibitional damage is previously unreported and may apply to other species with physiologies that make them difficult to store.
[show abstract][hide abstract] ABSTRACT: Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.
[show abstract][hide abstract] ABSTRACT: Embryonic axes of tea (desiccation sensitive) and pea (desiccation tolerant) were dried at different rates or stored at different water contents to distinguish between damage associated with the immediate effects of water loss and the longer-term effects of a partially hydrated state. No loss of viability was observed if pea axes were dried sufficiently rapidly (from 1.8 to 0.1 g H2O g-1dry mass (g/g) within 5 d). However, viability was lost in tea axes dried below 0.5 g/g (approximately -15 MPa) even if axes were dried within 1 h. Death in tea axes dried to moisture contents less than 0.5 g/g probably resulted from the removal of water necessary for cellular structural integrity (i.e. desiccation damage sensu stricto). When axes of both species were dried at slower rates, viability losses were observed at water potentials between about -3 and -15 MPa. The timing for this type of damage was species dependent, occurring within 2 d for tea and after 5 d for pea, and may be explained by higher oxidative activity in tea compared to pea. Embryos of both species with water potentials below -3 MPa were lethally damaged if oxygen consumption exceeded 1000–5000 μmol O2 g-1dry mass. Recalcitrant seeds are different than orthodox seeds because the former do not survive drying below a critical water content, regardless of the drying rate. Rapid drying is required for accurate assessment of the critical water content. Slow drying leads to metabolic imbalance and artefactual assessment of the critical water content for desiccation damage. Both tea and pea seeds were susceptible to damage from metabolic imbalances, suggesting that the predominant stress from slow drying is ageing.
[show abstract][hide abstract] ABSTRACT: This study investigated the interactions among water content, rapid (nonequilibrium) cooling to -196 degrees C using isopentane or subcooled nitrogen, and survival of embryonic axes of Aesculus hippocastanum. Average cooling rates in either cryogen did not exceed 60 degrees C s(-1) for axes containing more than 1.0 g H(2)O g(-1)dw (g g(-1)). Partial dehydration below 0.5 g gg(-1) facilitated faster cooling, averaging about 200 and 580 degrees C s(-1) in subcooled nitrogen and isopentane, respectively. The combination of partial drying and rapid cooling led to increased survival and reduced cellular damage in axes. Electrolyte leakage was 10-fold higher from fully hydrated axes cooled in either cryogen than from control axes that were not cooled. Drying of axes to 0.5 g g(-1), reduced electrolyte leakage of cryopreserved axes to levels similar to those of control material. Axis survival was assayed by germination in vitro. Axes with water contents greater than 1.0 g g(-1), did not survive cryogenic cooling. Between 1.0 and 0.75 g g(-1), axes survived cryogenic exposure but developed abnormally. The proportion of axes developing normally after being cooled in isopentane increased with increasing dehydration below 0.75 g g(-1), reaching a maximum between 0.5 and 0.25 g g(-1) after being cooled at > or =300 degrees C s(-1). Cooling rates attained in subcooled nitrogen did not exceed 250 degrees C s(-1), and normal development of axes was observed only at < or =0.4 g g(-1). These results support the hypothesis that rapid cooling enhances the feasibility of cryopreservation of desiccation-sensitive embryonic axes by increasing the upper limit of allowable water contents and overall survival.
[show abstract][hide abstract] ABSTRACT: Changes in ultrastructural, biochemical and biophysical characteristics of embryonic axes of Aesculus hippocastanum during development are related to changing levels of desiccation tolerance. Histodifferentiation was complete 30 days after flowering (DAF) and fruits were shed about 120 DAF. During this period, the dry mass of embryonic axes increased from about 0.5 to 4 mg and the water content decreased from 10.2 to 2.0 g H2O g-1 dry mass (g g-1). In spite of the large changes in water content, water potentials of freshly harvested material declined slightly during development from -0.65 to -2.0 MPa. Tolerance of desiccation increased as embryos matured. If evaluated on the basis of critical water contents for survival, tolerance appeared to increase continuously, maximum tolerance being achieved at 120 DAF when embryos survived water contents as low as 0.30 g g-1. When evaluated from critical water potentials, three distinct levels of desiccation tolerance could be recognized at -1.8 MPa (30-40 DAF), -4 M Pa (48-90 DAF) and -12 MPs (100-120 DAF). During development, total dry matter increased while sugar content (g g-1' dry mass) and osmotically active material (mmol g-1 dry mass) decreased. The subcellular organisation of axes was always typical of metabolically active tissues. Maximum tolerance (-12 MPa) was associated with a reduced amount of monosaccharides and the appearance of water with unusual calorimetric behaviour. Our data are consistent with several of the current hypotheses regarding the mechanisms of desiccation tolerance. Accumulation of dry matter reserves, reduced levels of monosaccharides, presence of dehydrin-like proteins and ability to form glasses appear to be associated with the changes in desiccation tolerance. However, none of these factors allow A. hippocastanum embryos to achieve the extreme level of desiccation tolerance typical of orthodox seeds. This may be because A. hippocastanum embryos do not reach physiological maturity and remain metabolically active even after they are shed from the parent plant. Also, embryos may acquire incompetent protectants or lack as yet unidentified protective mechanisms.
Physiologia Plantarum 11/1998; · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Germplasm must be stored under optimal conditions to maximize longevity and efficiently maintain genetic resources. In order to identify optimal storage conditions, we investigated the effects of temperature (−5 to 45°C) and water content (<0.17 g H2O g−1 dry weight) on longevity of Typha latifolia L. pollen. Longevity was highest at water contents corresponding to storage relative humidity (RH) of 11-15% which corresponded to the shoulder of water sorption isotherms. Also coinciding with this shoulder were abrupt changes in heat capacity of water present in the pollen. Consistent with changes in isotherms with temperature and the concept of critical RH for storage, optimum water contents increased with decreasing temperature. An attempt was made to explain the aging behavior according to the glass concept. The water content-temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. We also found a change in activation energy of aging in Arrhenius plots around Tg, demonstrating a change in aging kinetics when the glassy state is lost. We concluL that Tg curves cannot be used solely to predict precise conditions of optimum storage, but might be useful for predictions of storage longevity above optimum water contents. The data imply that too much drying reduces longevity and should be avoided, particularly when cryogenic storage is considered.
Physiologia Plantarum 05/1998; 103(2):145 - 153. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Water contents, desiccation tolerance, respiratory rates and subcellular characteristics of three contrasting seed types were studied during development. Avicennia marina (a tropical wetland species) and Aesculus hippocastanum (a temperate species) produce recalcitrant seeds and Phaseolus vulgaris produces orthodox seeds. During development, A. hippocastanum and P. vulgaris seeds showed a decline in water content and respiration rate with a concomitant increase in desiccation tolerance. These parameters did not change during the development of A. marina seeds once they had become germinable. There was a decrease in the degree of vacuolation and an increase in the deposition of insoluble reserves in A. hippocastanum and P. vulgaris seeds, while A. marina seeds remained highly vacuolated and did not accumulate insoluble reserves. Mitochondria and endomembranes degenerated during the development of A. hippocastanum and P. vulgaris seeds, but remained unchanged in A. marina seeds. The data are consistent with the hypothesis that extensive vacuolation and high metabolic rates contribute to desiccation sensitivity. However, the development of recalcitrant A. hippocastanum seeds is similar to that of orthodox P. vulgaris seeds. These data are in accord with the concept of seed recalcitrance being a consequence of truncated development. The results suggest that there may be three categories of seeds: orthodox seeds which develop desiccation tolerance, seeds which show similar development to orthodox seeds, but are shed before desiccation tolerance is well developed, and seeds which show no developmental trends giving rise to increased tolerance.
Seed Science Research 01/1997; 7:135-144. · 1.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 g water per g dry mass is believed to increase cytoplasmic viscosity. Cooling to -196 degree C was done at rates averaging between 0.17 and 1300 degree C per second, and warming at 600 or 1.35 degree C per second. Survival was assessed after 4 weeks in vitro. Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70 percent normal development ranged between 0.17 and about 1300 degree C per second for axes at a water content of 0.26 g water per g dry mass narrowing with increasing hydration to an apparent optimum at about 686 degree C per second in axes at 0.8 g water per g dry mass At 1.7 g water per g dry mass, axes cooled at 0.17 degree C per second yielded nearly 40 percent normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.