Desiccation Storage Research Articles

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

Heat Resistance, Virulence, and Gene Expression of Desiccation-Adapted Salmonella Enteritidis During Long-Term Storage in Low-Water Activity Foods.

Desiccation stress could induce crossprotection and even affect virulence of Salmonella enterica. However, the influence of food matrices with low-water activity on desiccation adaptation of Salmonella still remains unclear. This study investigated the survival and adaptation of Salmonella Enteritidis in skim milk powder, ginger powder, and chocolate powder under desiccation storage conditions for a total of 12 weeks. High survival rates of Salmonella Enteritidis in all food matrices maintained over the long-term desiccation storage. Desiccation-adapted Salmonella Enteritidis enhanced heat resistance (p < 0.05) with the increase of storage time. Food composition plays an important role in the induction of crossresistance of desiccation-adapted Salmonella. After desiccation storage, Salmonella Enteritidis in ginger powder was most tolerant to heat treatment. Salmonella Enteritidis in skim milk powder was most resistant to the gastrointestinal simulation environment, and had strongest adhesion to Caco-2 cells. The effects of food composition on gene expression (rpoS, proV, otsA, otsB, grpE, dnaK, rpoH, and sigDE) in desiccation-adapted Salmonella Enteritidis were not significant (p > 0.05). At initial desiccation storage, osmotic protection-related genes (fadA, proV, otsA, and otsB), stress response regulator (rpoS), and heat-resistance-related genes (grpE, dnaK, and rpoH) were all significantly upregulated (p < 0.05). However, after 4-week storage, the expression level of desiccation-related genes, proV, otsA, otsB, grpE, dnaK, and rpoH, significantly decreased (p < 0.05). This study enables a better understanding of Salmonella's responses to long-term desiccation stress in different kinds of low-water activity foods.

Metabolomics-based response of Salmonella to desiccation stress and skimmed milk powder storage.

The strong survival ability of Salmonella in low-moisture foods (LMFs) has been of public concern, and is considered a threat to people's health. Recently, the development of omics technology has promoted research on the molecular mechanisms of the desiccation stress response of pathogenic bacteria. However, multiple analytical aspects related to their physiological characteristics remain unclear. We explored the physiological metabolism changes of S. enterica Enteritidis exposed to a 24 h-desiccation treatment and a subsequent 3-month desiccation storage in skimmed milk powder (SMP) with an approach of gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-Q Exactive-mass spectrometry (UPLC-QE-MS). A total of 8,292 peaks were extracted, of which 381 were detected by GC-MS and 7,911 peaks were identified by LC-MS/MS, respectively. Through analyses of differentially expressed metabolites (DEMs) and key pathways, a total of 58 DEMs emerged from the 24 h-desiccation treatment, which exhibited the highest relevance for five metabolic pathways, involving glycine, serine, and threonine metabolism, pyrimidine metabolism, purine metabolism, vitamin B6 metabolism, and pentose phosphate pathway. After 3-month SMP storage, 120 DEMs were identified, which were related to several regulatory pathways including arginine and proline metabolism, serine and threonine metabolism, β-alanine metabolism, glycerolipid metabolism, and glycolysis. The analyses of key enzyme activities of XOD, PK, and G6PDH and ATP content provided further evidence that supported the metabolic responses such as nucleic acid degradation, glycolysis, and ATP production played an important role in Salmonella's adaptation to desiccation stress. This study enables a better understanding of metabolomics-based responses of Salmonella at the initial stage of desiccation stress and the following long-term adaptive stage. Meanwhile, the identified discriminative metabolic pathways may serve as potentially useful targets in developing strategies for the control and prevention of desiccation-adapted Salmonella in LMFs.

Enhancing long-term storage and stability of engineered living materials through desiccant storage and trehalose treatment.

Engineered living materials (ELMs) have broad applications for enabling on-demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast-laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1-year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ-CT and confocal microscopy indicate that cells form spherical colonies within F127-BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on-demand, cold-chain independent bioproduction.

Effect of storage time on the physiological characteristics and vegetative regeneration of desiccation‐tolerant mosses on the Loess Plateau, China

The artificial cultivation of moss biocrusts can accelerate the recovery of degraded arid lands and is closely related to moss productivity. Understanding the properties of inoculation materials on the regenerative capacity of mosses, in particular the effect of time limits on storage, has the potential to benefit the cultivation of artificial moss biocrusts. We investigated the vegetative regeneration and physiological characteristics of three desiccation‐tolerant mosses (Barbula unguiculata, Didymodon vinealis, and Didymodon tectorum) upon rehydration after periods of desiccation storage for 40, 89, 127, and 197 days. Regenerative capacity, represented by gametophyte vigor index, decreased with increased storage time. The greatest change in vegetative regeneration among storage times was observed in B. unguiculata, where the gametophyte vigor index decreased by 95.74% after 197 days of storage. Over the same period, there were smaller decreases in gametophyte vigor index of D. vinealis and D. tectorum of 42.17% and 13.30%, respectively. Malondialdehyde and soluble sugar increased with longer periods of storage time, while soluble protein content first increased, then decreased. Oxidation levels are important factors influencing the recovery of desiccation‐tolerant mosses. All three moss species regenerated after 197 days of storage, but regenerative capacity is dependent on species. For land managers, biocrust restoration can be facilitated by careful screening of suitable moss species, based on their capacity to regenerate new growth after extended periods of storage and selection of species based on variations in physiological characteristics.

Osmotic damage as a predictor of motility loss during convective desiccation of bovine sperm.

Current state-of-the art technologies are lagging in the application of desiccation storage to mammalian cells using nonreducing sugars. For bovine sperm, motility is irreversibly lost before reaching a sufficiently low moisture content necessary for preservation. It is hypothesized that much of the damage during drying is related to the osmotic stress encountered due to increased osmolarity of the extracellular environment. To test this hypothesis, we subjected sperm to liquid hyperosmotic environments for varying time-periods and measured their motility. We then extracted parameters for two models for motility loss based on these experiments: a first-order rate injury model (Fast or Slow) and a multi-modal (MM) injury model. The MM injury model incorporated an additional function accounting for damage induced by a time-independent osmotic change. Based on these models, we predicted sperm motility loss measured from natural and forced convective desiccation experiments. The MM injury model was able to closely bracket motility loss for desiccation as an osmotic change event with time-independent and time-dependent components. While the mechanistic basis of osmotic damage requires further exploration, the model can serve as a bracketing tool for predicting motility loss during desiccation based on excipients designed to minimize osmotic damage.

Buoyancy Driven Mass Transfer in a Liquid Desiccant Storage Tank

A new concept for long-term solar thermal storage is based on the absorption properties of aqueous calcium chloride. Water, diluted and concentrated calcium chloride solutions are stored in a single tank. An immersed heat exchanger and stratification manifold are used to preserve long-term sorption storage, and to achieve thermal stratification. The feasibility of sensible heating the tank via large-scale natural convection without mixing salt solutions is demonstrated via measurements of velocity, CaCl2 mass fraction, and temperature in a 1500 l prototype tank. Experiments are conducted over a practical range of the relevant dimensionless parameters. For Rayleigh numbers from 3.4 × 108 to 5.6 × 1010 and buoyancy ratios from 0.8 to 46.2, measured Sherwood numbers are 11 ± 2 to 62 ± 9, and the tank is thermally stratified. Convective mixing between salt layers is inhibited by the presence of a steep density gradient at the interface between regions of differing mass fraction. The predicted storage time scales based on mixing via natural convection for the reported Sherwood numbers are 160–902 days.

Mass transfer during sensible charging of a hybrid absorption/sensible storage tank

A concept for long term thermochemical storage based on absorption of aqueous CaCl2 is described and evaluated during sensible charging operation. The novelty of the desiccant storage tank is that strong and diluted salt solutions as well as water are stored in one vessel. An immersed heat exchanger and manifold provide the means to charge the tank without mixing solutions of different CaCl2 mass fraction. The ability to heat the tank via natural convection, while minimizing mass transfer between regions of different salt mass fraction, is elucidated via optical measurements of the velocity and CaCl2 mass fraction distributions in a 1500 liter prototype tank. Over a wide range of the dimensionless parameters that govern mixing, the mass transfer between layers of differing CaCl2 mass fraction is low, typically with Sherwood numbers less than 100, and temperature stratification is maintained. Projected time scales for long term storage exceed 100 days.

Comparison of frozen versus desiccated reference human red blood cells for hemagglutination assays

Red blood cells (RBCs) are commonly used fresh or stored in frozen format for identification of patients' antibodies and serologic specificity of such antibodies at reference laboratories. However, maintaining a large pool of fresh RBCs is impossible in a blood-banking environment and blood in frozen format poses a logistic disadvantage in terms of accessibility, maintenance cost, safety, and sample recovery. This study explores an alternative, desiccation storage method for RBCs to provide a reagent that supports greater utilization and flexibility for reference laboratories. RBCs from five donors were used in the study. RBCs were processed and kept in either frozen or desiccated format. Study variables for either the frozen or the desiccated cells included cell recovery as quantified by cell counts, gross microscopic examination, and hemagglutination assays. The mean percentage of cell recovery for thawed and washed frozen RBCs was 20% versus 50% for rehydrated and washed desiccated RBCs. Microscopic examination of thawed cells from the frozen preparation showed cells with irregular shapes, a sharp contrast when compared with rehydrated cells from the desiccated preparation, where cells are mostly intact, smooth surface, and biconcave in structure. Cells in both preparations performed well in manual agglutination tests. Desiccation preservation of RBCs provides a somewhat better RBC recovery and cell structure stability, while maintaining the necessary antigen-antibody reactions for cell surface markers, which will allow desiccated RBCs to be archived in blood collecting and processing reference laboratories.

Investigation on a two-stage solar liquid-desiccant (LiBr) dehumidification system assisted by CaCl 2 solution

A two-stage solar powered liquid-desiccant dehumidification system, for which two kinds of desiccant solution (lithium chloride and calcium bromide) are fed to the two dehumidification stages separately, has been studied. In the studied system air moisture (latent) load is separately removed by a pre-dehumidifier using cheap calcium chloride (CaCl 2) and a main dehumidifier using stable lithium bromide (LiBr). Side-effect of mixing heat rejected during dehumidification process is considerably alleviated by an indirect evaporative cooling unit added between the two dehumidification stages. The feasibility of high-desiccant concentration difference achieved by reusing desiccant solution to dehumidify air and regenerating desiccant repeatedly is analyzed. By increasing desiccant concentration difference, desiccant storage capacity is effectively explored. It is found that the pre-dehumidification effect of CaCl 2 solution is significant in high ambient humidity condition. Also seen is that the desiccant investment can be decreased by 53%, though the cost of equipments is somewhat increased, and the Tcop and COP of the proposed system can reach 0.97 and 2.13, respectively.

Seed longevity – moisture content relationships in hermetic and open storage

In seed storage research, moisture content can be maintained by providing a stable relative humidity (e.g. over saturated salt solutions) or by hermetic storage, but the two approaches provide different gaseous environments which might affect longevity. Seeds of timothy (Phleum pratense L.) and sesame (Sesamum indicum L.) were stored at 45 degrees C or 50 degrees C, respectively, with different moisture contents maintained by hermetic storage in laminated-aluminium-foil packets, or by desiccators above either saturated salt solutions or moistened silica gel. Seeds were withdrawn from storage at intervals from 1 to 28 d for up to 480 d and viability estimated. Within a species, the negative logarithmic relation between seed longevity and moisture content did not differ (P> 0.25, timothy; >0.05, sesame) between storage in desiccators over either moistened silica gel or saturated salt solutions, whereas the relation was much steeper (P< 0.005) in hermetic storage: longevity was similar at high moisture contents, but at low values much greater with hermetic storage. This effect of storage method on seed longevity's sensitivity to moisture content implies that oxygen is relatively more deleterious to seeds at lower than at greater moisture contents and confirms that hermetic storage is preferable for long-term seed storage at low moisture contents.

Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l−1 sorbitol, 20 g l−1 sucrose, 400 mg l−1 glutamine and 2 g l−1 Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l−1 sucrose, 400 mg l−1 glutamine, 1.5 g l−1 activated charcoal and 2 g l−1 Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l− sucrose, 20 g l−1 Phytagel, 1.5 g l−1 activated charcoal, 115.6 µM gibberellic acid and 214.8 µM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.

Involvement of Acetaldehyde in Seed Deterioration of Some Recalcitrant Woody Species through the Acceleration of Aerobic Respiration

The rate of acetaldehyde (Ald) evolution in the deterioration of recalcitrant woody seeds was investigated. Four plant species, Ligustrum japonicum, Quercus serrata, Quercus myrsinaefolia and Camellia japonica, were used for the experiments. Similar to orthodox seeds, all of the recalcitrant seeds used contained Ald in addition to methanol and ethanol, although the amount of Ald in Camellia, a typical oil seed, was very small. These volatiles were accumulated in a container in which Ligustrum and Q. serrata seeds were stored for a short period. Moreover, all of the seeds that had been previously exposed to Ald for only 6 d at 3 or 13 degrees C lost their vigor rapidly in proportion to the concentration of Ald. The occasional removal by decompression of Ald accumulated in the container prolonged the life span of Q. serrata seeds from 4 to 6 months. These findings suggest that a short life span of the hydrated recalcitrant seeds may involve Ald synthesis as in the orthodox seeds. However, the action mechanism of Ald in Ligustrum and Quercus seeds in which storage substances were polysaccharides seems to differ slightly from that in orthodox seeds, because their aerobic respiration was significantly stimulated by exposure to exogenously applied Ald. It was, therefore, thought that the rapid deterioration of some recalcitrant seeds in woody species may result from a decline in vigor, not only due to the denaturation of functional proteins by Ald as in the orthodox seeds but also due to the rapid consumption of direct substrates for the Ald-stimulated aerobic respiration and related co-enzymes within seeds. In contrast, in the oil-bearing Camellia seeds, Ald was slightly produced and their aerobic respiration was not enhanced by Ald, although they were very sensitive to Ald. Desiccation storage of Camellia seeds caused the deterioration of their outer part, which was accelerated by exogenously applied Ald, which suggests that in Camellia Ald acts only to denature the functional proteins as in orthodox seeds. Thus, the short longevity of these woody recalcitrant seeds is discussed in relation to the actions of Ald produced endogenously.

Long-term stabilisation potential of poly(vinylpyrrolidone) for amorphous lactose in spray-dried composites.

The aim of this study was to investigate the potential of poly(vinylpyrrolidone) (PVP) to inhibit the crystallisation of amorphous lactose during storage of the composites up to 6 months. Short-term stability was assessed by microcalorimetry over 10 days and long-term stability by storage in desiccators with different relative humidities for 3 and 6 months. The solid-state structure of the particles after storage was analysed by differential scanning calorimetry. It was found that the presence of PVP increased the critical relative humidity (RH) for crystallisation relative to the pure lactose and both the proportion and the molecular weight of the PVP affected the stabilisation of the amorphous phase. The difference in critical RH between the materials increased over time. The T(g) of the materials was generally reduced due to the absorption of water and it is suggested that the inhibiting effect therefore is related mainly to a specific interaction between lactose and PVP, rather than to a counteracting effect of the polymer on the moisture induced depression of T(g).

Impact of the host cadaver on survival and infectivity of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) under desiccating conditions.

Entomopathogenic nematode species of Steinernema carpocapsae, Steinernema riobrave, or Heterorhabditis bacteriophora were used to compare survival and infectivity among infective juveniles (IJs) emerging in water from hosts in White traps (treatment a), emerging in sand from hosts placed in sand (treatment c), and emerging from hosts placed on a mesh suspended over sand (treatment m). Nematode survival and infectivity was recorded in sand at three-day intervals during 21 days of storage in desiccators at 75% relative humidity and 25 degrees C. Infectivity was measured by exposing 5 Galleria mellonella for 16 h to IJs. Treatment did not affect percent survival of H. bacteriophora IJs. Percent survival of S. riobrave and S. carpocapsae IJs was lowest in treatment a. Across all treatments, by 10 days after the beginning of the experiments, IJ survival declined to 93, 43, and 28% of levels on day 1 for H. bacteriophora, S. riobrave, and S. carpocapsae, respectively. For the three treatments, infection rate over time was described by a negative exponential function for S. riobrave and S. carpocapsae and by a sigmoid function for H. bacteriophora.

Dry weight carbon and nitrogen content of some euphausiids from the north Atlantic Ocean and the Celtic Sea

Euphausiids were identified, measured and then dried during a research cruise to sites in the north-eastern Atlantic Ocean and shelf waters of the Celtic Sea in late summer. The lengths of furcilia larvae and later stages were measured in three ways, and regressions relating each pair of measures were calculated for different developmental stages of each species. Four hundred and ninety-three dried specimens of nine species were weighed and length-weight regressions were calcu- lated. A subset of 187 specimens was selected for analysis of carbon (C) and nitrogen (N) content soon after return to the laboratory. The C and N contents of a further 122 specimens were determined after the specimens had been stored for 9 years. No consistent differences in percentage C and N content were found between the two groups, indicating no systematic change during prolonged storage in desiccators. Calyptopes of Nyctiphanes couchi contained a lower percentage of both C (mean 36.2%) and N (mean 8.6%) than later stages (means: 40.6% C, 11.0% N). Carbon content and C:N ratios of a group of specimens of Thysanoessa longicaudata, including some juveniles, adult males and adult females, were distinctly higher (>46% and >5.0, respectively) than those of the majority of specimens of the same species.

Desiccant solar air conditioning in tropical climates: I—Dynamic experimental and numerical studies of silicagel and activated alumina

This paper presents a dynamic study of moist air dehumidification in view of its use in an air conditioning process by evaporative cooling in tropical climates. A special device has been built to study dehumidification of tropical-like inlet air, through a fixed compact bed of silicagel and activated alumina. The compact desiccant storage is composed of two parallel beds to reduce the pressure drop. A good agreement is obtained for our experiment, and the computed amount of cycled water from the numerical model in the adiabatic process. This analytical model is used to simulate a complete air conditioning open cycle operating with hot and humid air.

Germinability of sclerotia of Macrophomina phaseolina

The germination of sclerotia of Macrophomina phaseolina from 3- to 20-day-old PDA cultures incubated at 32 °C and 24 °C varied from 98 to 25% and 75 to 30%, respectively. Germinability of sclerotia from cultures of all ages tested was rapidly lost during storage in desiccators for 24 h at 6 °C or in screw-top vials at room temperature for 30 days. Germination of sclerotia stored in screw-top vials at 6 °C for 30 days declined less rapidly. There was a linear relationship between the size of the sclerotium and the number of germ tubes emerging from it. The role of sclerotium size in survival and what factors may be responsible for loss of germinability with increasing age of source culture are discussed.

Hybrid Tea-rose pollen. I. Germination and storage

Germination and storage trials were carried out with pollen of several rose varieties. The pollen grains germinated well in a 15% sucrose solution with 40 ppm boric acid. Staining the pollen with a 0.1% tetrazolium solution and standardizing the degree of colour at which the pollen grains are counted as viable, provided a good viability estimate, simpler to carry out than in vitro germination. Germination capacity and staining ability of the pollen were greatly impeded-about halved-by dehydration during storage in desiccators at low humidity. This effect could be corrected by humidifying the pollen beforehand for about one hour, though this pre-treatment increased the percentage of germinated pollen grains more than the percentage stained. There was no difference between the two percentages in fresh or in deep-frozen pollen. Pollen stored at 1°C and high relative humidity soon lost its germination capacity: between 0 and 20% humidity a considerable proportion of the pollen remained viable for 9 months and longer. Storage for the same period in vacuum-sealed glass tubes at −24°C maintained viability as well or better and would probably prolong it further. Some of the cold-stored pollen induced a reasonable seed set after one year, a low seed set was obtained even after two years of storage at 1°C and low humidity.

Storage effect following treatment of barley seeds with ethyl methanesulfonate—I. Influence of seed moisture content

Barley seeds were treated with ethyl methanesulfonate (EMS) for 3 hr at 25°C, then washed with tap water for 24 hr at 25°C, redried at 40°C to different moisture contents, and stored at 25°C. The different seed moistures, attained by redrying, were maintained during storage in desiccators with different H 2 O/H 2 SO 4 solutions. The criteria used for expressing the effect of storage were chromosomal aberrations, M 1 germination, M 1 survival, M 1 seed set, and the frequency of M 2 -chlorophyll mutants. During the 1-6 weeks storage three types of response to the seed moisture level were observed: (1) Following storage of EMS-treated seeds with 30 per cent moisture, the biological damage in the M 1 generation as well as the M 2 -chlorophyll mutation frequency decreased considerably, as compared with treatments without storage; (2) Following storage of EMS-treated seeds with 20 and 13 per cent moisture, the biological damage in the M 1 generation increased considerably, so that, in the field experiments, no plants survived; (3) Following storage of EMS-treated seeds with 5 per cent moisture, the biological damage in the M 1 generation and the M 2 -chlorophyll mutation frequency remained unchanged, as compared with treatments where no storage was applied.