Similarity in the effects of different seed cryoconservation conditions on plant growth and development

Morphological traits of 35 wild plant species were studied after freezing the seeds using quick or programmed two-stage freezing to ?196°C and nondeep freezing to ?10°C. The seeds were stored frozen for 1 month. The seeds of all studied species had low moisture content. Field and laboratory germination rates, leaf growth, the number and length of shoots, the number of generative organs, and variation of all these traits were studied. It was demonstrated that the traits changed in the same directions independently of the freezing conditions, except for laboratory germination rate for some species. The changes were determined by the species-specific plant characteristics rather than by the freezing mode.     

Effect of freezing of seeds on the morphological characteristics of four pink species

The effect of non-deep freezing (-10 degrees C) and deep freezing (-196 degrees C) on the morphological characteristics of four pink species was investigated. It was shown that the growth and development of plants after cryopreservation changed insignificantly. Rather stable characteristics (diameter of flower, the number of cauline nodes, and root length) did not change. The length of generative shoots decreased a little, and the character of histograms changed. On the other hand, the changeable characteristics (the number of vegetative shoots and flowers) decreased after seed cryopreservation. However, the number of flowers did not vary in the next year. The parameters essential for seed cryopreservation, seed germination and the quantity of fruits, did not changed. It was also found that seed freezing exerts a stimulating effect on seed germination and the top limit of some parameters, including the quantity of generative organs.     

Effects of elevated temperature and carbon dioxide on seed-set and yield of kidney bean (Phaseolus vulgaris L.)

It is important to quantify and understand the consequences of elevated temperature and carbon dioxide (CO₂) on reproductive processes and yield to develop suitable agronomic or genetic management for future climates. The objectives of this research work were (a) to quantify the effects of elevated temperature and CO₂ on photosynthesis, pollen production, pollen viability, seed‐set, seed number, seeds per pod, seed size, seed yield and dry matter production of kidney bean and (b) to determine if deleterious effects of high temperature on reproductive processes and yield could be compensated by enhanced photosynthesis at elevated CO₂ levels. Red kidney bean cv. Montcalm was grown in controlled environments at day/night temperatures ranging from 28/18 to 40/30 °C under ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ levels. There were strong negative relations between temperature over a range of 28/18–40/30 °C and seed‐set (slope, ? 6.5% °C^?1) and seed number per pod (? 0.34 °C^?1) under both ambient and elevated CO₂ levels. Exposure to temperature > 28/18 °C also reduced photosynthesis (? 0.3 and ? 0.9 µmol m^?2 s^?1 °C^?1), seed number (? 2.3 and ? 3.3 °C^?1) and seed yield (? 1.1 and ? 1.5 g plant^?1 °C^?1), at both the CO₂ levels (ambient and elevated, respectively). Reduced seed‐set and seed number at high temperatures was primarily owing to decreased pollen production and pollen viability. Elevated CO₂ did not affect seed size but temperature > 31/21 °C linearly reduced seed size by 0.07 g °C^?1. Elevated CO₂ increased photosynthesis and seed yield by approximately 50 and 24%, respectively. There was no beneficial interaction of CO₂ and temperature, and CO₂ enrichment did not offset the negative effects of high temperatures on reproductive processes and yield. In conclusion, even with beneficial effects of CO₂ enrichment, yield losses owing to high temperature (> 34/24 °C) are likely to occur, particularly if high temperatures coincide with sensitive stages of reproductive development.     

Influence of frost on nutrient resorption during leaf senescence in a mangrove at its latitudinal limit of distribution

The latitudinal distribution of mangrove species is limited mainly by low temperature. Leaf scorch and massive leaf fall are the predominant symptoms of frost damage. Nutrient resorption during leaf senescence is an important adaptation mechanism of mangroves. Abnormal defoliation disturbs nutrient resorption. We evaluated the effects of frost on nutrient loss of mangroves and the protective effects of warmer seawater inundation on reducing nutrient loss. On January 14, 2009, the most cold-tolerant mangrove Kandelia obovata at its naturally latitudinal limit (Fuding, China, 27°17??N) was exposed to freezing temperature (?2.4°C) for 4 h (minimum ?2.8°C). The freezing air temperature occurred during flood tide, resulting that the flooded shoots were protected by warmer seawater. Frost caused 31.3% and 13.0% defoliation on the exposed shoots and the flooded shoots, respectively. Frost restricted nutrient resorption during leaf senescence. K. obovata resorbed 61% N and 42% P during normal leaf senescence, respectively. However, frost-damaged leaves only resorbed 13% N and 10% P during the course, respectively. Foliar N:P molar ratios were <31, suggesting N limitation. Tidal inundation can partially protect mangroves from frost damage. Reduced nutrient resorption efficiency and massive leaf fall caused by frost add pressure to mangroves under nutrient limitation at their latitudinal limits.     

Biological activity of Friend spleen focus-forming virus after cold storage

Two stocks of Friend spleen focus-forming virus (SFFV) were prepared, one in saline and the other in Eagle's medium with 2% fetal calf serum, and the effects of different freezing, storage and thawing temperatures were determined for the recovery of infectious virus from each diluent. Once frozen, virus maintained its titer at ?70 and at ?170 °C for up to 13 weeks, while it lost titer at ?13 °C more rapidly if it had been prepared in saline than in medium. However, during the freezing process lower ambient temperatures (?70 and ?170 °C) gave lower virus yields than a higher temperature (?13 °C) did. Similarly, rapid thawing (in a 37 °C water bath) was less efficient than slow thawing (in 4 or 20 °C air) for the recovery of infectious SFFV, This study illustrates the importance, for efficient recovery of leukemogenic activity from stored murine leukemia virus stocks, of the temperature used for freezing or thawing, as well as for storage.     

Effects of freeze-thaw stress on bacterial populations in soil microcosms

To test the effect of freezing on soil biota, isolated from the shortgrass prairie of northeastern Colorado, a series of experiments were performed using gnotobiotic soil microcosms.Pseudomonas paucimobilis was used to examine the effects of freezing on bacteria of different growth stages. Secondly, the effect of multiple freeze-thaw cycles was tested on an assemblage of bacterial species. Lastly, the effect of freezing on predator-prey interactions was studied usingP. paucimobilis and an amoebal predator,Acanthamoeba polyphaga. A temperature of ?9°C was not detrimental toP. paucimobilis at any growth stage. A single severe freeze-thaw cycle (?27°C to 23°C) resulted in 40–60% mortality ofP. paucimobilis and the mixed bacteria, although additional freezing events did not reduce the populations further. Multiple freeze-thaw cycles (?9°C to 23°C) gave 40–60% mortality ofP. paucimobilis and the mixed bacteria. Predator-prey population cycles were possibly desynchronized by freeze-thaw events.     

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance

To test the hypothesis that the up‐regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild‐type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over‐expressing sucrose phosphate synthase (over‐sps) or with antisense repression of either cytosolic fructose‐1,6‐bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over‐expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over‐sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (?9.1 and ?7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (? 5.2 and ?5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over‐sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over‐wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.     

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium

Leaflets of Sphagnum capillifolium were exposed to temperatures from ?5°C to +60°C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at ?1.1°C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 μm indicates a cell volume reduction of approximately ?82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (?1.1°C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (?16.1°C; LT₅₀) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5°C which is significantly below the heat tolerance of chlorophyllous cells (49.9°C; LT₅₀). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.     

Anthesis and seed production in Zostera marina L. (eelgrass) from the chesepeak by

Anthesis and seed production in Zostera marina L. were studied in three areas of the Chesapeake Bay from January to June 1980. Inflorescence primordia with distinguishable anthers and pistils were first observed in February when water temperature was 3°C. Development of the reproductive shoots in the field continued after February as water temperature rose, with the first evidence of pollen release in mid-April (water temperature 14.3°C). Stigmata loss was first observed in samples taken in late April at all locations by as water temperatures averaged above 16°C. Pollination was complete at all locations by 19 May and anthers were no longer present. Few reproduction shoots were found on 3–5 June and seed release was assumed to be complete by this time (water temperature 25°C). The density of flowering shoots ranged from 11 to 19% of the total number of shoots, producing an estimated 8127 seeds m^?2.Comparison of flowering events with other areas along a latitudinal gradient from North Carolina to Canada indicated that reproductive events occurred earlier in the most southern locations and at successively later dates with increasing latitude.     

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion‐weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high‐temperature exotherm (?3°C to ?5°C) corresponded with ice formation and high ice‐nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (?6°C and ?8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low‐temperature exotherm corresponded with SAM injury and the killing temperature in wheat (?21°C) and rye (?27°C). The SAM had lower ice‐nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.     

Divergent seed production responses of white and blue flowers of Gentiana leucomelaena (Gentianaceae) to warming and watering

Background: The adaptation, evolution and function of flower colour diversity in response to changing environments are one of the oldest puzzles in plant ecology. It is logical that comparative studies on fitness of flower colour would be the most meaningful if they were conducted on the same genotype.

Aims: We used Gentiana leucomelaena, which produces both white and blue flowers on different tillers of the same individual, as a model species to test adaption and fitness differences of contrasting flowers in contrasting environments.

Methods: We examined seed production by white and blue flowers in response to increased temperatures (28 °C/2 °C vs. 12 °C/2 °C; 12 h light/12 h dark) in a climate chamber study and to watering in the field (+1.5 l m^?2 d^?1 vs. control with no supplementary irrigation).

Results: For white and blue flowers warming decreased but watering increased seed number. Increased temperature and watering increased the size of seeds from white flowers, but neither warming nor watering significantly changed seed size of blue flowers. Seed size was significantly negatively correlated with seed number in the temperature treatments but positively correlated in watering treatments. The positive correlation was strong in white flowers, but the negative correlation was stronger in blue than in white flowers.

Conclusions: Water availability and low temperature confer an advantage to white flowers, while warming and dry habitats favour blue flowers. These divergent responses may influence total plant fitness and thus help explain the adaptive value and evolution of flower colour diversity.     

Does cross‐acclimation between drought and freezing stress persist over ecologically relevant time spans? A test using the grass Poa pratensis

• Despite evidence that prior exposure to drought can increase subsequent plant freezing tolerance, few studies have explored such interactions over ecologically relevant time spans. We examined the combined effects of drought and subsequent freezing on tiller growth and leaf sugar concentrations in the grass, Poa pratensis .
• We exposed tillers to no drought (?0.04 MP a), moderate drought (?0.19 MP a) or severe drought (?0.42 MP a) for 3 weeks in summer. Tillers were then frozen in autumn or spring at ?5 °C (frost damage) or at 0 °C (control) for 3 days and harvested after a re‐growth period.
• For shoot growth, there was a significant interaction between drought and autumn freezing, whereby the relative effect of freezing on growth was least for the plants previously exposed to severe drought; however, there was no significant interaction between drought and spring freezing. For root growth, there were no significant interactions between drought and freezing in either season. Leaf sugar concentrations increased significantly with drought intensity, but these effects dissipated within a month, prior to the onset of the autumn freezing treatment.
• Overall, our results suggest that interactions between prior drought and subsequent freezing in P. pratensis may be most relevant in the context of autumn freezing, and despite the important role of soluble sugars in increasing both drought and freezing tolerance in this species, the retention of these compounds after drought stress does not appear to explain the occurrence of drought–frost interactions at ecologically relevant time scales.

     

Altitudinal variations in germination and growth responses ofReynoutria japonica populations on Mt Fuji to a controlled thermal environment

The authors examined altitudinal variations in the thermal responses of seed germination and seedling growth inReynoutria japonica (=Polygonum cuspidatum) under controlled environmental conditions. Seed populations were collected from different altitudes on Mt Fuji in Japan. The mean seed weight of the upland populations (above 1500 m) was significantly (1.5-fold) heavier than that of the lowland populations (below 1400 m). Under the lowest temperature regime of 15/10°C (day/night) the upland populations showed a significantly higher percentage and speed of germination than the lowland populations; this was not significant under higher temperature regimes. These results indicate that the germination traits of the upland populations on Mt Fuji are favorable for colonization in their cold habitats (low temperature and short growing season). Growth and shoot development were compared between the seedlings grown from seeds collected at altitudes of 700 and 2420 m. The upland seedlings showed a significantly larger biomass and leaf area than the lowland seedlings at 15°C, but there was no difference at 25°C. The difference in biomass at 15°C was attributed to the difference in seed weight. The upland seedlings produced a significantly larger number of branches with smaller and more numerous leaves at both 15°C and 25°C. these developmental traits of the upland seedlings were considered to represent the adaptation of the life form to upland environments. It was concluded that theR. japonica populations along an altitudinal gradient on Mt Fuji can be classified into two ecotypes, whose distribution border lies at an altitude of about 1400–1500m. In this study, the seed weight and germination traits of twoR. japonica seed populations collected in Chiba Prefecture were briefly compared with those of the lowland populations on Mt Fuji.     

How endangered is sexual reproduction of high-mountain plants by summer frosts? Frost resistance, frequency of frost events and risk assessment

In temperate-zone mountains, summer frosts usually occur during unpredictable cold spells with snow-falls. Earlier studies have shown that vegetative aboveground organs of most high-mountain plants tolerate extracellular ice in the active state. However, little is known about the impact of frost on reproductive development and reproductive success. In common plant species from the European Alps (Cerastium uniflorum, Loiseleuria procumbens, Ranunculus glacialis, Rhododendron ferrugineum, Saxifraga bryoides, S. moschata, S. caesia), differing in growth form, altitudinal distribution and phenology, frost resistance of reproductive and vegetative shoots was assessed in different reproductive stages. Intact plants were exposed to simulated night frosts between ?2 and ?14 °C in temperature-controlled freezers. Nucleation temperatures, freezing damage and subsequent reproductive success (fruit and seed set, seed germination) were determined. During all reproductive stages, reproductive shoots were significantly less frost resistant than vegetative shoots (mean difference for LT₅₀ ?4.2 ± 2.7 K). In most species, reproductive shoots were ice tolerant before bolting and during fruiting (mean LT₅₀ ?7 and ?5.7 °C), but were ice sensitive during bolting and anthesis (mean LT₅₀ around ?4 °C). Only R. glacialis remained ice tolerant during all reproductive stages. Frost injury in reproductive shoots usually led to full fruit loss. Reproductive success of frost-treated but undamaged shoots did not differ significantly from control values. Assessing the frost damage risk on the basis of summer frost frequency and frost resistance shows that, in the alpine zone, low-statured species are rarely endangered as long as they are protected by snow. The situation is different in the subnival and nival zone, where frost-sensitive reproductive shoots may become frost damaged even when covered by snow. Unprotected individuals are at high risk of suffering from frost damage, particularly at higher elevations. It appears that ice tolerance in reproductive structures is an advantage but not an absolute precondition for colonizing high altitudes with frequent frost events.     

Cryopreservation of some useful microalgae species for biotechnological exploitation

Culture collections of microalgae represent a biological resource for scientific research and biotechnological applications. When compared to the current methods of maintenance and sub-culturing, cryopreservation minimizes labor costs and is an effective method for maintaining a large range of species over long periods with high stability. In order to determine the best cryopreservation method for microalgae species with great biotechnological potential, three freezing protocols were employed using different cryoprotectants (dimethyl sulfoxide—Me₂SO; methanol—MeOH). Three marine microalgae species (Thalassiosira weissflogii; Nannochloropsis oculata, and Skeletonema sp.) were cooled by directly plunging into liquid nitrogen (?196°C) and with two-step controlled cooling protocols (?18°C and ?80°C pre-treatments). After storage periods ranging from 10 to 120 days, viability was determined by the ability of cells to actively grow again. Results obtained for T. weissflogii showed that this species could be preserved at ultra-low temperature (?196°C) for 10 and 30 days with 10?% Me₂SO and 5?% MeOH when employed a controlled cooling protocol (?80°C). N. oculata was successfully cryopreserved either by direct freezing or with controlled cooling protocols. N. oculata samples presented good responses when treated with 5?% Me₂SO, 10?% Me₂SO, 5?% MeOH and even without any cryoprotectant. Skeletonema sp. did not survive cryopreservation in any of the tested conditions. The results indicate the difficulty in establishing common protocols for different microalgae species, being necessary further studies for a better understanding of cell damages during freezing and thawing conditions for each species.     

Effects of freezing to −196 °C and thawing on Setaria lutescens seeds

The effects of single and repeated freezing and thawing of Setaria lutescens seeds in liquid nitrogen were investigated. One freeze to ?196 °C followed by a slow thaw, increased seed germination from 40 to 70%, but additional freeze-thaw cycles reduced germination to 30%. Using a scanning electron microscope, evidence was produced that seed coat cracking did not cause either initial increased, or subsequent reduced germination. Observations with a transmission electron microscope revealed that disruption of the integrity of lipid bodies accompanied increased damage from repeated freezing at ?196 °C and thawing. Repeated freezing and thawing of seeds stored in liquid nitrogen should be done with care to avoid loss of the germplasm.     

Freezing tolerance of chickpea: biochemical and molecular changes at vegetative stage

The aim of this study was to find a correlation between the freezing tolerance of three chickpea (Cicer arietinum L.) cultivars (?nci, I??k-05, and Sar?-98) and their wild relative C. echinospermum and physiological responses. Chickpea plants (15-d-old) were subjected to cold acclimation (CA) (10 °C for 7 d), freezing (-3 or -5 °C for 2 h), and subsequent rewarming (25 °C for 7 d). In two separate experiments with three replications, we determined growth, water status, photosystem 2 photochemical activity, photosynthetic pigments, H₂O₂, malondialdehyde, and proline content, relative leakage ratio, antioxidant enzyme activities, and gene expressions in cultivars different in freezing tolerance. Freezing temperatures adversely affected all the physiological parameters of all cultivars. Rewarming did not lead to complete recovery. The cultivar ?nci was more tolerant to the freezing temperatures than others.     

Polypeptide markers for low temperature stress during seed germination in sunflower

Sunflower seeds behaved as chilling and freezing sensitive and also exhibited acclimation under low seed moisture content (< 1 %). At high seed moisture content (approx. 22 %) they tolerated chilling stress but failed to acclimate under freezing temperatures. Pre-imbibitional chilling (5 °C) or freezing (−5 or −10 °C) stress significantly enhanced total soluble protein (TSP) content. Chilling treatment after imbibition (in contrast to pre-imbibition) enhanced germination and this was accompanied by increase in 30, 24 and 21.9 kDa TSPs content (3 d after germination). Freezing at −5 and −10 °C suppressed seed germination and increased content of 78 and 56.2 kDa wall bound proteins. Chilling acclimation decreased 35.4, 33.9, 29.5, 23.4 and 21.4 kDa TSPs.     

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (?23 °C) or not. Two mutant lines of rice callus when frozen to ?196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.