ForMAX - a beamline for multiscale and multimodal structural characterization of hierarchical materials.

The ForMAX beamline at the MAX IV Laboratory provides multiscale and multimodal structural characterization of hierarchical materials in the nanometre to millimetre range by combining small- and wide-angle X-ray scattering with full-field microtomography. The modular design of the beamline is optimized for easy switching between different experimental modalities. The beamline has a special focus on the development of novel fibrous materials from forest resources, but it is also well suited for studies within, for example, food science and biomedical research.

Cryopreservation of In Vitro-Grown Shoot Tips of the Medicinal Species Cleome Spinosa (Cleomaceae) Applying Vitrification-Based Techniques.

BACKGROUND: Few cryopreservation studies have been reported with the genus Cleome. Due to the use of C. spinosa in traditional medicine and its valuable pharmacological potential, the long-term conservation of the species will allow the safe maintenance of its germplasm. OBJECTIVE: This study compares two vitrification-based techniques on the cryopreservation of shoot tips of C. spinosa. MATERIALS AND METHODS: The effect of sucrose preculture and different vitrification solutions was evaluated using vitrification and V Cryo-plate techniques. The supplementation of recovery medium with BAP was also assessed. RESULTS: The V Cryo-plate proved to be the most efficient technique. Treatment of shoot tips with PVS2 at 0°C resulted in a higher regeneration response after cryopreservation when compared to treatment with PVS2 and PVS3 at 25°C. The highest survival (83.3%) and recovery (76.6%) were achieved for shoot tips exposed to PVS2 for 90 min at 0°C and recovered on MS medium supplemented with 0.5 mg L-1 BAP for 2 weeks. CONCLUSION: Plants regenerated from cryopreserved shoot tips maintained their in vitro multiplication capacity and showed a normal phenotypic aspect, demonstrating the efficiency of the cryopreservation protocol.

Recovery of Oil Palm (Elaeis guineensis Jacq.) Somatic Embryos Cryostored For 20 Years.

　　BACKGROUND: A cryopreservation protocol has been established for oil palm somatic embryos (SEs), the efficiency of which must be evaluated, both in terms of regeneration and of long-term storage capacity, before its large-scale routine use. OBJECTIVE: To test the survival and recovery of 29 clones of oil palm somatic embryos cryostored for 20 years. MATERIALS AND METHODS: Clumps of SEs were pregrown for 7 days on medium containing 0.75 M sucrose, dehydrated in air-tight containers containing silica gel to moisture contents between 19-35% fresh weight, and then immersed directly in liquid nitrogen and stored in cryotanks for 20 years. RESULTS: Survival of SEs cryopreserved and rewarmed immediately displayed an average value of 19.1% for the 29 clones tested while survival of SEs rewarmed after 20 years of cryostorage was significantly higher, with an average of 33.2% for the 28 surviving clones. Out of these 28 surviving clones, three were lost due to contamination or regrowth decline, six produced only shoots and the rest proliferated. CONCLUSION: It is possible to cryostore oil palm SEs for extended periods and to regenerate proliferating cultures and plantlets from the cryopreserved material. The cryopreservation protocol established can thus be efficiently used to store oil palm germplasm and to manage large-scale production in industrial laboratories.

Cryopreservation of Arecanut (Areca catechu L.) Pollen.

BACKGROUND: Cryopreservation opens new avenues in the field of genetic resource conservation, especially in recalcitrant seeded palms such as arecanut for which field genebanks are exposed to pest and disease attacks and natural calamities. It is only through cryopreservation that the safety of the conserved germplasm can be assured at a relatively low cost for extended periods. OBJECTIVE: The objective of this work was to standardize various aspects of arecanut pollen cryopreservation, viz. collection and desiccation of pollen, in vitro germination, viability and fecundity studies. MATERIALS AND METHODS: Pollens of three arecanut genotypes (Sumangala, Hirehalli Dwarf and Hirehalli Dwarf x Sumangala) were collected in December 2013-February 2014. In vitro viability tests were conducted using fresh and desiccated pollen. Desiccated pollen was cryopreserved by direct immersion in liquid nitrogen and cryostored for different durations (24 hours to 2 years). Viability and fertility studies were conducted using cryopreserved pollen. RESULTS: Pollen extraction was achieved from fully opened male flowers by desiccation at room temperature (33-34 degree C). A medium containing 2.5 g/L sucrose was found to be best for in vitro germination at room temperature. There was no significant difference in germination between desiccated and cryopreserved pollen whereas pollen tube length decreased significantly after cryopreservation. Fertility studies using HD x Sumangala pollen cryostored for various durations (1 month, 1 year and 2 years) showed the setting of 70, 43 and 62%, respectively. Normal nut set was observed using cryopreserved pollen. CONCLUSION: Pollen cryopreservation is a viable option for germplasm conservation and hybridization programmes in arecanut.

Impact of irradiation and immunosuppressive agents on immune system homeostasis in rhesus macaques.

In this study we examined the effects of non-myeloablative total body irradiation (TBI) in combination with immunosuppressive chemotherapy on immune homeostasis in rhesus macaques. Our results show that the administration of cyclosporin A or tacrolimus without radiotherapy did not result in lymphopenia. The addition of TBI to the regimen resulted in lymphopenia as well as alterations in the memory/naive ratio following reconstitution of lymphocyte populations. Dendritic cell (DC) numbers in whole blood were largely unaffected, while the monocyte population was altered by immunosuppressive treatment. Irradiation also resulted in increased levels of circulating cytokines and chemokines that correlated with T cell proliferative bursts and with the shift towards memory T cells. We also report that anti-thymocyte globulin (ATG) treatment and CD3 immunotoxin administration resulted in a selective and rapid depletion of naive CD4 and CD8 T cells and increased frequency of memory T cells. We also examined the impact of these treatments on reactivation of latent simian varicella virus (SVV) infection as a model of varicella zoster virus (VZV) infection of humans. None of the treatments resulted in overt SVV reactivation; however, select animals had transient increases in SVV-specific T cell responses following immunosuppression, suggestive of subclinical reactivation. Overall, we provide detailed observations into immune modulation by TBI and chemotherapeutic agents in rhesus macaques, an important research model of human disease.

Coconut (Cocos nucifera l.) pollen cryopreservation.

BACKGROUND: Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. OBJECTIVE: The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. MATERIALS AND METHODS: Pollen of two coconut varieties (West Coast Tall WWCTW and Chowghat Orange Dwarf CODC) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. RESULTS: Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32 % in desiccated pollen whereas it was between 26 and 29 % in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31 % in desiccated pollen, while it was between 28 and 32 % in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 nm and 226-396 mm for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1 % after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively. CONCLUSION: These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

Phenotypic and molecular characterization of plants regenerated from non-cryopreserved and cryopreserved wild Solanum lycopersicum mill. Seeds.

BACKGROUND: Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. OBJECTIVE: In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. METHODS: Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. RESULT: No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. CONCLUSION: Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.

Moderate alcohol consumption enhances vaccine-induced responses in rhesus macaques.

We have recently shown that chronic alcohol consumption in a rhesus macaque model of ethanol self-administration significantly modulates the serum cytokine profile. In this study, we extended these observations by investigating the impact of chronic ethanol exposure on the immune response to Modified Vaccinia Ankara (MVA). All animals were vaccinated with MVA before ethanol exposure to ethanol and then again after 7 months of 22 h/day of "open-access" drinking of 4% (w/v) ethanol. Our results indicate that animals whose blood ethanol concentration (BEC) chronically exceeded 80 mg/dl had lower CD4 and CD8 T cell proliferation as well as IgG responses following MVA booster than control animals. In contrast, relatively moderate drinkers whose BEC remained below 80 mg/ml exhibited more robust MVA-specific IgG and CD8 T cell responses than controls. To begin to uncover mechanisms underlying the differences in MVA-specific responses between the three groups, we analyzed plasma cytokine levels and microRNA expression in peripheral blood mononuclear cells following MVA booster. Our findings suggest that moderate ethanol consumption results in higher levels of antiviral cytokines and an expression profile of microRNAs linked to CD8 T cell differentiation. In summary, moderate alcohol consumption enhances recall vaccine responses, whereas chronic alcohol intoxication suppresses this response.

Biochemical characterization of ecuadorian wild Solanum lycopersicum Mill. plants produced from non-cryopreserved and cryopreserved seeds.

This paper presents some of the effects of cryopreservation of wild Solanum lycopersicum Mill. seeds on the early stages of germination post liquid nitrogen exposure. Percentage of germination, conversion into plantlets and plant fresh mass were evaluated after cryostorage. Levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and proteins were determined. Peroxidase and superoxide dismutase activities were recorded. Liquid nitrogen exposure increased the percentage of seed germination at 5 days but at 7 days, the conversion into plantlets and the plant fresh mass were not statistically different between non-cryopreserved and cryopreserved samples. Several significant effects of cryopreservation were recorded at the biochemical level at 7 days of germination under controlled conditions. Highly significant effects due to liquid nitrogen exposure were observed in leaves: increased levels of peroxidase enzymatic and specific activities and cell wall-linked phenolics. Very remarkable effects were also recorded in roots: decreased contents of chlorophylls and cell wall-linked phenolics.

Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plates.

Two cryopreservation procedures using aluminium cryo-plates, termed V-Cryo-plate and D-Cryo-plate, were successfully developed for in vitro mat rush (Juncus decipiens Nakai) basal stem buds. Multiple stems induced in liquid MS medium containing 8.9 µM BA by roller culture were cut into small clumps, plated on solid MS medium and cultured for 1 week at 25 degree C. Clumps that had produced many buds were cold-hardened at 5 degree C for 1-2 months. The buds with basal stems were dissected from small clumps and precultured overnight at 25 degree C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in loading solution (2 M glycerol + 1.0 M sucrose). In the D-Cryo-plate procedure, the buds were dehydrated to 27-25% moisture content (fresh weight) by placing the cryo-plates in the air current of a laminar flow cabinet for 2 to 3 h. In the V-Cryo-plate procedure, buds were dehydrated by immersing the cryo-plates in PVS2 vitrification solution for 40 min at 25 degree C. In both procedures, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in medium with 1.0 M sucrose for 20 min at room temperature. Regrowth of cryopreserved buds of line 'Kitakei 2' using D-Cryo-plate and V-Cryo-plate procedures, was 90% and 80%, respectively. The two procedures were applied to 20 additional mat rush lines. Using the V-Cryo-plate procedure resulted in regrowth ranging between 13.3 and 86.7%, with an average of 52.5%. The D-Cryo-plate led to regrowth ranging between 73.3 and 96.7%, with an average of 86.3%. The D-Cryo-plate procedure will facilitate cryostorage of mat rush germplasm.

Cryopreservation of Arachis pintoi (leguminosae) somatic embryos.

In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (Nakai) hairy roots using a systematic approach.

A systematic approach using a set of 13 treatments was applied to develop a droplet-vitrification protocol for Rubia akane hairy roots, based on their responses to preculture, loading, dehydration and cooling/rewarming steps. The roots were very sensitive to osmotic stress induced by both preculture in liquid sucrose-enriched medium (up to 0.5 M sucrose) and by dehydration with highly concentrated vitrification solutions (VSs). Loading was necessary before dehydration of explants with VS, and the composition of the loading solution (LS) significantly affected their post-cryopreservation regeneration. Due to high sensitivity of roots to both chemical cytotoxicity and osmotic stress produced by VSs, cryoprotection with alternative VSs, i.e. B5-80 percent (40 percent glycerol + 40 percent sucrose, w/v) at room temperature for 15 min or with A3-70 percent (29.2 percent glycerol + 11.7 percent DMSO + 11.7 percent EG + 17.4 percent sucrose, w/v) at 0 degree C for 20 min ensured the highest post-cryopreservation regeneration. However, when using these solutions, endothermic peaks (enthalpies) with -2.9 and -5.8 J per gram fresh weight, respectively, were recorded by differential scanning calorimetry (DSC) during the rewarming phase. Droplet-vitrification using foil strips showed higher post-cryopreservation regeneration (86 percent) compared with vitrification in cryovials (59 percent), possibly due to the higher cooling and rewarming rates achieved with droplet-vitrification. The developed protocol was applied to hairy roots of five other species with minor modifications in explant type, the duration of the last subculture before explant excision, and the dehydration duration with VS B5-80 percent.

Cryobanking of Korean allium germplasm collections: results from a 10 year experience.

This paper reviews a 10-year experience in establishing a cryopreserved Allium germplasm collection at the genebank of the National Agrobiodiversity Center, Republic of Korea. A systematic approach to Allium cryopreservation included: 1. revealing the most critical factors that affected regeneration after cryostorage; 2. understanding the mechanisms of cryoprotection by analyzing the thermal behavior of explants and cryoprotectant solutions using DSC and influx/efflux of cryoprotectants using HPLC; 3. assessing genetic stability of regenerants; and 4. revealing the efficiency of cryotherapy. Bulbil primordia, i.e. asexual bulbs formed on unripe inflorescences, proved to be the most suitable material for conservation of bolting varieties due to high post-cryopreservation regrowth and lower microbial infection level, followed by apical shoot apices from single bulbs and cloves. A total of 1,158 accessions of garlic as well as some Allium species have been cryopreserved during 2005-2010 using the droplet-vitrification technique with a mean regeneration percentage of 65.9 percent after cryostorage. These results open the door for large-scale implementation of cryostorage and for simplifying international exchange for clonal Allium germplasm.

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification.

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

Development of alternative loading solutions in droplet-vitrification procedures.

In plant vitrification protocols, the loading treatment, which involves treating the explants with a moderately concentrated cryoprotectant solution, precedes dehydration of explants with highly concentrated vitrification solutions in order to reduce the toxicity which can be induced by their direct exposure to such highly concentrated solutions. This study aimed at developing alternative loading solutions composed of mixtures of glycerol and sucrose at various concentrations. Differential scanning calorimetry runs of loading solutions and of loaded and dehydrated explants were performed to assay thermal events occurring during cooling and warming. These loading solutions were applied to two model species, viz. garlic and chrysanthemum which were cryopreserved using a droplet-vitrification procedure. The loading treatment proved to be beneficial to both garlic and chrysanthemum and increased recovery of cryopreserved explants. However, response to the loading solutions tested varied between the two model species employed: with garlic, all the loading solutions had a similar effect, whereas survival of chrysanthemum shoot tips was significantly influenced by the composition of the loading solution employed. A loading solution comprising 1.9 M glycerol and 0.5 M sucrose was the most effective. The loading treatment may thus act as an osmotic stress neutralizer and/or induce the physiological adaptation of tissues and cells, including membranes, to both dehydration and freezing.

Field development of oil palms (Elaeis guineensis jacq) originating from cryopreserved stabilized polyembryonic cultures.

In this paper, the long term observation of plants originating from control and cryopreserved stabilized polyembryonic cultures (SPCs) of six elite oil palm clones was carried out. Survival of plantlets in the nursery was monitored, then a series of vegetative and floral characteristics of over 440 palms were studied for up to 12 years after field transfer in Côte D'Ivoire. The six clones tested showed an average recovery of 34% after freezing in liquid nitrogen. The average survival in the nursery of plantlets originating from pretreated and dehydrated and from cryopreserved SPCs was higher than that of control SPCs. Palm trees originating from control SPCs were found to flower earlier than those originating from pretreated and dehydrated and from cryopreserved SPCs. This delay in flowering disappeared progressively and all palms had flowered 3 years after planting regardless of the SPC treatment. Abnormal palms were observed in one clone only. With this clone, the percentage of abnormal palms originating from cryopreserved SPCs was significantly lower (5%) than that measured on palms originating from control SPCs (29%).

Cryopreservation of seeds and in vitro-cultured protocorms of Oncidium bifolium sims. (Orchidaceae) by encapsulation-dehydration.

Encapsulation-dehydration was employed for cryopreserving seeds and in vitro-cultured protocorms of Oncidium bifolium. Freshly harvested seeds, 120 days after pollination, were encapsulated in beads containing 1/2 MS medium with 3% sucrose and 3% calcium alginate and subsequently pretreated in agitated (80 rpm) liquid medium supplemented with 0.15 M sucrose (24 h) followed by 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h). The beads with seeds were dehydrated with silica gel for 5 h to 19.2% moisture content and immersed in liquid nitrogen for 1 h, thawed at 30 degrees C for 2 min, post-treated using the same series of liquid media [0.5 M sucrose (24 h), 0.25 M sucrose (48 h), 0.15 M sucrose (24 h)], and recultured on 1/2 MS medium with 0.1M sucrose and 0.7% percent agar. As much as 4.8% of the cryopreserved seeds produced complete plants. In-vitro cultured protocorms were successfully cryopreserved following the same procedure, allowing 11.3% of them to produce plants.

Cryopreservation of potato cultivated varieties and wild species: critical factors in droplet vitrification.

The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40 degrees C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%.

New determinants for tolerance of coffee (Coffea arabica L.) seeds to liquid nitrogen exposure.

The present work establishes for the first time that tolerance of coffee seeds to liquid nitrogen (LN) exposure depends on the initial quality of the seedlot and on the rewarming regime employed. Seedlot quality was estimated by the parameters of a quantal response model of desiccation sensitivity developed previously. The percentage of seedlings recovered from cryopreserved seeds was very well correlated with the relative humidity (RH) at which 90 percent of the initial viability was retained, RH90, as estimated by the model. Whatever the cooling regime employed, rewarming the seeds slowly by exposing them to ambient air was highly detrimental. Slow rewarming-induced viability loss was not due to imbibitional damage since seeds pre-heated at 37 degree C after slow rewarming to 0 degree C exhibited a survival percentage lower than seeds thawed rapidly to 0 degree C before sowing. The optimal hydration status for coffee seed cryopreservation was also re-examined. Drying seeds in 81 percent RH provided survival percentages considerably higher than those obtained using the drying RH always employed until now, i.e. 78 percent. A new procedure for slowly precooling the seeds prior to immersion in LN was also established. It consisted of placing the vials containing the seeds in a dry ice-bath for 25 min. Using this procedure in combination with seed drying in 81 percent RH and rapid rewarming in a 37 degree C water-bath for 30 min ensured the highest survival percentages ever obtained with coffee seeds, i.e. 89 percent, a value which was not significantly different from the initial viability percentage.

Optimal hydration status for cryopreservation of intermediate oily seeds: Citrus as a case study.

BACKGROUND AND AIMS: The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily seeds using Citrus as a model. METHODS: The relationships between equilibrium relative humidity (RH), seed water content, presence of freezable water as determined by DSC analysis, and germination percentage after immersion in liquid nitrogen (LN) were investigated in Citrus aurantifolia, C. grandis, C. madurensis and C. reticulata. The relationship between the lipid content of seeds and their unfrozen water content was also investigated. KEY RESULTS: Independent of their level of seed desiccation tolerance, the optimal desiccation RH for seed tolerance to LN exposure was 75-80 % in the four species studied. This optimal hydration status always coincided with that at which presence of frozen water could not be detected in seed tissues during the cooling/thawing process. The unfrozen water content of seeds was variable between species and negatively correlated to seed lipid content. Using the present data, those obtained previously in seven coffee species and those reported by other authors for five other species, a significant linear relationship was found between the lipid content and the unfrozen water content of seeds. CONCLUSIONS: This study provides additional evidence that intermediate oily seeds do not withstand the presence of freezable water in their tissues during the cooling/warming process. Moreover, it offers two important applied perspectives: (1) independent of their level of desiccation tolerance, testing germination of seeds of a given oily seed species after equilibration in 75-80 % RH at 25 degrees C and LN exposure, gives a rapid and reliable evaluation of the possibility of cryopreserving whole seeds of this given species; (2) it is now possible to calculate the interval of water contents in which non-orthodox oily seeds of a given species are likely to withstand LN exposure as a function of their lipid content.