Concentrations Of EMS Research Articles

Effects of caffeine or EDTA post-treatment on EMS mutagenesis in soybean

Seeds of soybean cultivar LD4 were mutagenically treated with EMS (0.3, 0.5, 0.6, 0.9, 0.5 and 1.8%) for 3 h only or plus caffeine (50 mM) or EDTA (1 mM) post-treatment for 5 h. The experimental results indicated that: (1) of the different concentrations of EMS treatment, the M2 mutation frequency induced with 0.6% EMS was the highest (9.7%). When the EMS concentration was over 0.9%, the mutation frequency decreased rapidly. (2) Of the EMS treatments plus caffeine or EDTA post-treatment, the mutagenic effect of 0.6% EMS was the best for inducing morphological variations. Caffeine post-treatment decreased notably the mutation frequency of EMS treatment; when concentrations of EMS were very high (1.5% and 1.8%), mutation frequencies of EDTA post-treatment were still 5.0% and 4.88%, but no mutants were found in EMS treatment or plus caffeine post-treatment. (3) In the M2 mutation spectrum, 11 kinds of mutant types were observed in EMS treatment or plus caffeine or EDTA post-treatment. Relative frequencies of some mutant types (growth period, plant height, grain size, leaf shape and sterility, etc.) were similat among the three treatments, but EDTA post-treatment could change the relative frequencies of yield characteristics (number of pods and grains, grain weight/plant) induced by EMS treatment only.

Mutagenic Effects of Combined and Single Doses of Gamma Rays and EMS in Opium Poppy*

AbstractMutagenic effects of gamma rays and EMS using both single and combined dosages were examined in opium poppy (Papaver somniferum L.). For inducing both macro‐ and micro‐mutations in the present study, combined mutagenic doses, especially 5 kR + 0.4 % EMS appeared to be greatly superior to all single doses of the two mutagens: gamma rays and EMS. The lowest dose of combined mutagen (5 kR + 0.2 % EMS) was about twenty times more effective than the most effective dose (15 kR) of gamma rays and twice as effective as the most effective concentration (0.4 %) of EMS. The combined dose 5 kR + 0.4 % EMS enhanced morphine content (%) to 0.98 as against 0.64 in the control. In accordance with earlier findings (GAUL 1964) the study revealed a correlation between induced macro‐ and micro‐mutations.

Chromosomal analysis in mouse eggs fertilized in vitro with sperma exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS)

Chromosome aberrations were analyzed at the first-cleavage metaphase of mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) as well as to methyl and ethyl methanesulfonate (MMS and EMS). The frequencies of chromosome aberrations markedly increased with dose of UV as well as with concentration of MMS and EMS. In the UV-irradiation group, the frequency of chromosome-type aberrations was much higher than that of chromatid-tupe aberrations. About 90% of chromosome aberrations observed in the eggs following MMS and EMS treatment to sperm were chromosome type in which the frequency of chromosome fragments was the highest. The effects of UV on the induction of chromosome aberrations were clearly potentiated by post-treatment incubation of fertilized eggs in the presence of Ara-C or caffeine, but the effects of MMS and EMS were not pronounced by post-treatment of Ara-C or caffeine. The results indicate a possibility that UV damage induced in mouse sperm DNA is reparable in the eggs during the period between the entry of sperm into the egg cytoplasm and the first-cleavage metaphase.

Alkylating agents induced chromosomal aberrations in lentil (Lens culinaris Med. var. T. 36)

Dry and dormant seeds of lentil (Lens culinaris Med.) var.T.36 were soaked separately in varying concentrations of EMS, NMU and DES. Study of meiosis in plants raised from these seeds revealed that a high frequency of translocations leading to multivalent associations involving varying number of chromosomes were induced in all the treatments. In case of all the three mutagens percentage of PMCs showing meiotic anamolies increased with the increase in concentration upto a certain level but decreased above it. The effects of mutagens in enhancing the chromosomal aberrations were in the order: NMU>EMS>DES

Linear relationship between lethal mutation yield and intake of ethyl methanesulfonate in Drosophila melanogaster.

Males of Drosophila melanogaster were fed sucrose solutions containing various concentrations of EMS (from 0.25 to 10 mM) for 24 hr. To measure the intake of an EMS solution, 3H-labeled sucrose was added to the feeding solution, and the 3H activity inside the flies was used as a measure for the intake volume of EMS solution. Each absorbed dose of EMS was estimated from the intake volume of an EMS solution multiplied by its EMS concentration in the feeding solution in a way similar to that described in a previous report [Ayaki et al, 1984]. The relationship between the estimated absorbed dose and the exposure concentration was almost linear in a low concentration range but became concave with a downward curvature in a high concentration range. The dose-response relationship between the frequency of sex-linked recessive lethals and the estimated absorbed dose showed no deviation from linearity at all the five absorbed doses tested. It may be concluded that the absorbed doses thus estimated were very close to true absorbed doses, indicating the usefulness of the present method for dosimetry of chemicals to be given to flies.

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact. V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture. The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture. In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive. Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1−2 × 10 6 cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10 5 cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10 6 cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.

BIOLOGICAL EFFECTS OF MUTAGENIC TREATMENTS IN RICE

Experiments were carried out with rice variety Co.37 to study effects of gamma Irradiation and EMS on germination, survival, pollen and seed fertility in M, generation. A progressive reduction in germination and survival was observed as the doses of gamma rays and EMS wore increased. The reduction in germination percentage was more following combination treatments than individual treatments and the maximum reduction (10.86%) was observed (40 Krad + 50mM). The lower concentrations of EMS did not affect the survival of seedlings and the reduction in survival percentage was more due to gamma Irradiation and combination treatments than EMS. For the combination treatments, the effect was additive on germination and survival, Pollen and seed fertility was markedly reduced in gamma ray treatments compared to the EMS treatments. The combination treatments produced additive effect on pollen and seed fertility in My generation.

Gamma rays and ethyl methanesulfonate induced mitotic abnormalities in Turnera ulmifolia Linn. var. angustifolia Willd.

The present paper deals with the gamma radiation and EMS induced chromosomal aberrations in Turnera ulmifolia Linn. Mitosis was found to be quite normal in control plants. As a result of the mutagenic treatment of seeds (gamma rays and EMS), chromosomal aberrations like stickiness, fragments, precocious movement of chromosomes, mitotic bridges and non-orientation of chromosomes were induced in the root tip cells. These irregularities were more at higher exposure of gamma rays and higher EMS concentrations for longer duration. The probable causes of the production of mitotic abnormalities following seed treatment with the mutagenic agents are discussed. The paper also includes effect of the physical and chemical mutagen on the cell division in the present plant.

Mutation induction in mammalian cell cultures Dissection of the expression time into its genetical and physiological components

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1−2 × 106 cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 105 cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 106 cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.

Resting Skeletal Muscle Membrane Potential as an Index of Uremic Toxicity

Electrochemical disturbances of skeletal muscle cells in untreated uremia are characterized by an increase in the intracellular sodium and chloride content, a decrease in intracellular potassium, and a low resting membrane potential. In this study, we have reexamined the foregoing and, in addition, have examined the effects of hemodialysis. Three groups of patients were studied. In the first group of 22 uncomplicated uremic patients, whose creatinine clearance (Ccr) ranged from 2 to 12 cm(3)/min per 1.73 m(2), resting transmembrane potential difference (Em) of skeletal muscle cells was measured. In each of the nine patients whose Ccr ranged between 6.3 and 12 cm(3)/min, the Em was normal (i.e., -90.8+/-0.9 mV, mean+/-SEM). However, as Ccr dropped below 6.3 cm/min, the Em became progressively reduced and assumed a linear relationship with the Ccr. In the second study, nine individuals with end-stage renal disease, whose mean Ccr was 4.3 cm(3)/min, underwent measurement of Em and intracellular electrolyte concentration before and after 7 wk of hemodialysis. Before dialysis, the Em was -78.5+/-2.1 mV, intracellular sodium and chloride were elevated, and the intracellular potassium was reduced. After 7 wk of hemodialysis the Em rose to -87.8+/-1.3 mV, and the intracellular sodium, chloride, and potassium became normal. In the third study, seven patients who were stable on 6-h thrice-weekly dialysis were studied before and after reduction of dialysis to 6 h twice weekly. In those individuals whose Em remained normal after 6 wk, dialysis time was reduced further. On thrice-weekly dialysis the Em was -91.2+/-1.0 mV. With reduced dialysis, the Em fell to -80.1+/-0.8 mV (P < 0.001). In each case, the Em became abnormal before significant signs or symptoms of uremia were noted. These findings demonstrate that end-stage renal disease is associated with serious electrochemical changes in the muscle cell which are reversed by hemodialysis and recur when dialysis time is reduced. Thus, serial observations of muscle Em may be a potentially powerful tool to assess adequacy of dialysis therapy.

Effect of denervation and ouabain on the response of the resting membrane potential of rat skeletal muscle to potassium

The effects of denervation and ouabain on the relationship between resting membrane potential (EM) and extracellular potassium concentration were determined for rat soleus (SOL) and extensor digitorum longus (EDL) muscles. At concentrations above 20 mM for both SOL AND EDL fibres the relationship could be described adequately by the Nernst equation. Denervation resulted in reduction of EM and a decrease in the slope of the relationship between EM and potassium concentration. 10(-3) M ouabain produced the same effects as denervation. The results are discussed in relation to the alterations in membrane permeability previously shown to occur as a consequence of denervation. It is concluded that the low EM of denervated muscle is the result of an increase in membrane permeability to sodium.

Interspecific comparison of ethyl methanesulfonate-induced mutation rates in relation to genome size

A detailed presentation is made of the experimental data from the various systems used by Heddle and Athanasiou to conclude that “the relationship between mutation rate and genome size is as striking for EMS as it is for radiation, thus confirming the reality of the ABCW relation” and that “there is a relatively constant value of rec for all organisms.” It is noted that 2 of the 9 mutation rates cited came from seed treatments of higher plants and were not converted to haploid-genome mutation rates necessary for a valid comparison with other test systems. It is argued that the use of EMS-induced mutation rates from Drosophila and mouse post-meiotic male germ cells for comparisons with radiation-induced mutation rates from Drosophila and mouse spermatogonia is inappropriate. The paucity of the available data that was used is emphasized, and the dearth of all available data is recognized. The method of using the actual initially applied concentration of EMS alone as a measurement of “dose” is criticized. Nevertheless, when this procedure recommended by Heddle and Athanasiou as being necessary “to provide a consistent measure of dose” for inter-specific comparisons of EMS-induced mutation rates is applied, the papers from which the data they used were scrutinized, and other available mutation-rate data assessed, no evidence was found for a relationship between EMS-induced haploid-genome mutation rate and genome size in organisms from E. coli to H. vulgare that differ in DNA content by a factor of more than 1000. X-Ray-equivalent values differed by more than two orders of magnitude from one test system to another and by one order of magnitude for different male-germ cells of Drosophila. Our previous analysis of the available data for radiation-induced mutation rates found no relationship between mutation rate and genome size. It is considered that the failure to find a relation between EMS mutation rate and genome size from the type of analysis suggested by Heddle and Athanasiou has no bearing on the utility of the rec concept. Uncritical extrapolation from one chemical to another and to an X-ray equivalent of genetic damage has been forcefully criticized elsewhere.

Somatic mutations in Tradescantia stamen hairs exposed to ethyl methanesulfonate

Young inflorescences of Tradescantia (BNL clone 02) heterozygous for flower color were treated with 0 (water), 0.1 or 0.5% EMS solutions for 2, 4, 8 or 16 hr. The frequency of somatic pink mutations in the stamen hairs scored 10–18 days after treatments was higher with the higher concentration of EMS and increased with lengthening duration of the treatment. The mutation frequency was also found to be clearly a function of the EMS dose in %·hr. The frequency of mutation increases with EMS dose; on a log-log graph the slopes were 1.37 or 1.31, when the mutation frequency was expressed as the number of pink mutant events per 10 3 hairs or per 10 5 cell divisions, respectively. That the slopes are steeper than 1 suggests the involvement of two-break deletions, but the possibility of insufficient penetration of EMS in cases of shorter treatments cannot be ruled out. The mutation frequencies observed were somewhat comparable to those induced by 0.25 to 10 rad of sparsely ionizing radiations at various dose rates. (1 − 4, 6, 16, 17)

SPONTANEOUS AND ETHYL METHANESULFONATE-INDUCED MUTATIONS CONTROLLING VIABILITY IN DROSOPHILA MELANOGASTER. II. HOMOZYGOUS EFFECT OF POLYGENIC MUTATIONS

Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.

ON THE INTERPRETATION OF MUTAGENICALLY INDUCED MOSAICISM IN DROSOPHILA

This paper draws attention to the formal parallelism that exists between chromosomal-loss mosaicism and mutagenically induced mosaicism in Drosophila and suggests that, although the underlying processes by which these two types of genetic mosaics are generated are very different, the more refined methodology employed in developmental analyses of genetically induced chromosomal-loss mosaics may be profitably extrapolated to mutagenesis studies. Results obtained from various studies of genetically induced mosaics and from a previous EMS mutation induction experiment at the yellow locus are utilized to illustrate this methodology and to estimate the total mutagenicity rates of EMS.-The following are some of the tentative conclusions that have been drawn in this report regarding an EMS concentration that produced 31% F(2) lethals in the standard X-linked recessive lethal test: (1) The frequency of cuticular mosaics is at least 5 times that of F(3) lethals. (2) At least 60% of all cuticular mosaics go undetected in the standard X -linked recessive lethal test since their mutant tissue does not extend into the germ line. (3) The frequency of EMS-induced cryptic mosaics is probably less than 10% the frequency of cuticular mosaics. (4) Some EMS-induced mutations are probably bona fide completes; if confirmed, this inference must be taken into consideration in estimating the total mutagenicity rates of this agent and in molecular interpretations of its mechanism of action. (5) The fact that the proportion of mutant tissue in EMS-induced mosaics is greater than 25% is consistent with the suggestion that the action of EMS is occasionally delayed until after the first cleavage division of the embryo. (6) Such an EMS concentration causes on the average more than 5 independent genetic alterations in the entire haploid genome of an X-bearing sperm.-This report clarifies the experimental evidence that must be generated, and the methodology that can be used to analyze this evidence, if it is of interest to render these and related conclusions regarding the effect of EMS on D. melanogaster more accurate, or if it is of interest to conduct a similar analysis for other mutagens that cause a significant degree of mosaicism.

Granulocyte-Monocyte Colony-forming Capacity of Human Marrow: A Clinical Study

Abstract A modification of the in vitro culture of marrow cells in soft agar to form granulocyte-monocyte colonies has been applied to human marrow using a constant stimulus of colony-stimulating factor (CSF) in the form of pooled serum from endotoxin-treated C57BL mice (EMS). With this system reproducible increments in colony formation were observed between two concentrations of EMS. The results indicate that the incremental response to two doses of CSF (CSF response ratio) combined with an observation of the number of colonies formed provide a more reliable measurement of colony-forming cells (CFC) than colony counts at a single dose level of CSF. The CSF response ratio in 43 marrow cultures from 34 patients with no obvious disturbance in granulopoiesis was found to be 1.65 (± 0.17 SD). Hodgkin’s disease with normal marrow showed a mean CSF response ratio of 2.04. Two subjects recovering from drug-induced agranulocytosis showed increased CSF response at a time when the blood was still neutropenic and the marrow showed hyperplasia and immaturity of the granulocytic tissue. In contrast, patients with sustained neutropenia and impaired marrow granulopoiesis showed CSF response ratios of less than 1.65. CSF response ratios in chronic myelogenous leukemia averaged 1.72, often with extremely high numbers of colonies per plate. Patients with untreated acute leukemia and marrows replaced by blasts did not form colonies. However, with partial leukemic marrow replacement, lymphoblastic leukemia marrow formed more colonies than did that of myeloblastic leukemia.

Effectiveness and efficiency of single and combined treatments of radiations and ethyl methane sulphonate inSorghum

The effect of single treatments with X- and gamma rays and EMS and combination treatments of gamma rays sand EMS on survival, seedling growth and seed fertility in M1 and chlorophyll mutation frequency in M2 was studied in the variety Co. 18 ofS. subglabrascens. There was an increase in the reduction of survival and seedling height and seed fertility with an increase in the dose of mutagens. Combined treatments were found to be more effective in reducing the survival and height of seedlings than single treatments, while EMS caused more drastic effect on seed fertility than rest of the mutagens. The frequency of chlorophyll mutations in the M2 increased with dose upto a certain dose level and then levelled off at high doses. EMS induced higher frequency of chlorophyll mutations than treatments with radiations. Combination treatments were found to be the most efficient in inducing the chlorophyll mutations as compared to single treatments. Mutagenic effectiveness was found to be the maximum at high doses of gamma rays and EMS, whereas higher efficiency values were observed at lower concentrations of EMS and middle doses of radiations. EMS was found to be the most effective and efficient mutagen than rest of the mutagens.

Elastolytic enzymes and elastin in the aging rat. I. Pancreatic elastolytic enzymes.

Since improved methods make it possible to determine both elastoproteinase and elastomucase activity in crude pancreatic extracts, the effect of age on enzyme titer in the pancreas was studied in virgin Wistar rats of both sexes. In males, the elastoproteinase concentration (in elastoproteinase unit [EU] per gram acetone-dried pancreas) decreased with age over the whole period of 0–24 months; the elastomucase Em-I and Em-S concentrations (in elastomucase unit [Em-U] per gram acetone-dried pancreas) rapidly decreased with age up to 12 months (Em-S) and 18 months (Em-I), remaining nearly constant after that age period. In female rats, the elastoproteinase concentration remained constant during the 1st year of life, increased rapidly between 15 and 18 months, then decreasing to the 15-month level at an age of 27 months. The titer of both elastomucases fell during the growth of the females, remaining constant between 6 and 15 months of age and showed a rapid increase in enzyme level between 15 and 18 months. Although there was a tendency for the enzyme concentration to decrease after 18 months, the fall was not statistically significant. The data obtained with rat pancreas were compared with those for human pancreatic enzymes and for human serum ‘elastoproteinase’ reported previously. Similar age-relationships were found.

Effects of gamma irradiation and ethyl methane sulphonate on morphometric traits and prevalence of common viral diseases and whiteflies in cassava.

Cassava (Manihot esculenta Crantz) is an important staple and food security crop for millions of people in Africa. However, its nutritional value is limited; yet its productivity is constrained by several pests and diseases. Induced mutagenesis is one approach with the potential to overcome such biotic stresses. The objective of this study was to assess the variability in morphometric traits and prevalence of common viral diseases and whiteflies in cassava to different doses and concentrations of gamma irradiation and ethyl methane sulphonate (ems) treatments. The effects were assessed on seed germination and growth of stakes, as well as foliar viral disease symptoms and whitefly counts. Radio-sensitivity tests revealed LD50 for sprouting as 37.6Gy for g-irradiation and 0.08% for EMS treatment. There was notable decrease in sprout, epicotyl length, shoot height, petiole length and number of leaf lobes, with increasing g-ray doses and EMS concentrations. Contrastingly, total chlorophyll content increased with increasing doses of g-rays and EMS concentrations. Basing on foliar whitefly counts and disease symptoms, there was a general increase in susceptibility to whitefly infestation and cassava mosaic disease (CMD) incidence. Significantly varying levels of resistance or tolerance to whiteflies and CMD were observed among plantlets derived from irradiated and EMS treated stems, compared to the controls. These findings lay a foundation for more future research on breeding for various traits, including disease resistance in cassava using induced mutagenesis approach.

Variierung der ÄMS-wirkung bei gerste durch anwendung verschiedener behandlungs- und nachwaschtemperaturen

The aim of post washing is to reduce the after-effect of EMS. In order to understand the influence of post washing temperature, it appeared necessary to investigate first the effect of EMS-treatment temperature. Barley seeds were treated in a 3·6 per cent EMS-solution for 3 hr at various temperatures. All criteria of biological damage increased with increasing treatment temperature: reduction of seedling height, lethality, sterility and frequency of chlorophyll mutations. This result is attributed to an increased permeability of cell walls so that more EMS molecules are taken up, as well as a higher rate of reactivity of EMS, especially with the nucleophilic groups. The influence of different post washing temperatures was studied for one EMS-treatment temperature, namely 25°C. With increasing post washing time, seedling height, survival rate and fertility increased, at each of the temperatures used, whether the seeds were sown immediately or redried. The values for these criteria decrease, however, with increasing temperature of post washing, at each of the different post washing periods used when the seed were sown immediately. On redrying, different post washing temperatures result in maxima of seedling height which are characteristic for each post washing period and the same applies to the rate of survival. Redrying after a short period of post washing (6 and 12 hr), at each post washing temperature, leads to shorter seedlings than without redrying. However, through extended post washing (24 hr) at 20°C or at a higher temperature, the seedlings of the redried seeds are as tall or even taller; the values for survival and fertility are higher too. Neither the period of post washing, the post washing temperature, nor redrying had any influence on the frequency of chlorophyll mutations; except for the 24-hr post washing at 10°C, which gave lower mutation rates. In the discussion it is shown that time and temperature of post washing, as well as redrying, may lead to reactions which result partly in opposite effects. The results of these experiments can be explained by the following assumptions and findings. Increasing time of post washing diminishes the concentration of EMS and augments the sensitivity to the mutagen. Increasing temperature during post washing accelerates both processes. It also results in faster reaction of the residual EMS and its increased hydrolysis into ethyl alcohol and methane sulfonic acid. The toxicity of the products of hydrolysis plays an important part in the physilogical damage and sterility. Redrying produces an increase in the concentration of residual EMS and a desensitization of the cells (which is raised by pre-soaking). The high redrying temperature (40°C) exerts its influence only at the second phase of redrying, since at the first phase a reduced temperature results from evaporation. The agreement of these results with previously published ones shows again that through post washing and redrying of EMS-treated seeds the reproducibility of the results is improved. Proof is given also that through adequate post treatment manipulations, physiological damage and sterility on one hand and mutation rate on the other may vary independently of each other.