Inducing Chlorophyll Mutations Research Articles

Influence of respiratory inhibitors on ethyl methanesulphonate induced mutagenic effects in Hordeum vulgare.

With an object of modifying the treatment conditions based on the concept of selection sieves, which may result in the higher mutagenic effectiveness and efficiency, barley seeds were treated with ethyl methanesulphonate (EMS) in various com-bination with non-toxic concentration of sodium azide (NaN3) and sodium arsenite (NaAsO2) as chemical modifiers. Treatments were carried out with dry, watersoaked and dried back seeds. Effects of these chemical modifiers in respect of pre-, post- and during treatment of EMS were also investigated considering the parameters, seedling injury, chromosomal aberrations, chlorophyll chimeras and sterility in M1 and chlorophyll deficient mutations as an index in M2. NaAsO2 did not induce chlorophyll mutations. However, NaN3 even in aqueous non-buffered solution induced chlorophyll mutations. As regards to their modifying action, although sodium azide and sodium arsenite both were used as common respiratory inhibitors, NaN3 was found to be most effective as a pre-treatment modifier in case of dry and dry-back seed treatments. Treatment of NaN3 soaked dried seeds with EMS synergistically increased the mutation frequencies but in general, combination treatments of NaN3 and EMS with soaked seeds were found to be highly deleterious. NaAsO2 has not been fonud to be effective as a pre-treatment modifier. It was very effective in during and post- EMS treatment combinations.

Mutagenic response of rhodes grass ( Chloris gayana Kunth.) to gamma rays

In order to determine the sensitivity of Rhodes grass ( Chloris gayana Kunth.) to gamma rays and to find an optimum gamma ray dose for inducing mutations, the seeds of a local Rhodes grass cultivar were exposed to nine doses of gamma rays ranging from 10 to 90 krad. Germination, seedling growth, survival, plumule, radicle and seedling lengths, pollen fertility, and morphological variants in the M 1 generation and chlorophyll mutation frequency, and spectrum in the M 2 generation were studied. In general, there was a decrease in all biological criteria studied with an increase in dose of gamma rays in M 1 generation. A dose dependent response was observed (viz., germination, r = −0.93; seedling height, r = −0.99; survival, r = −0.98; and pollen fertility, r = −0.97) and at higher doses the effecr was drastic. The LD 50 values for all biological end points were relatively high except for pollen fertility. The morphological variants included: earhead proliferation, nodal variant, peduncle variant, leaf curl, grassy type, partial sterility, apical sterility, high sterility and gappy ears. In the M 2 generation, the chlorophyll mutation frequency increased in a linear fashion at low (10–30 krad) and medium (40–60 krad) doses and was erratic at higher doses (70–90 krad). The chlorophyll mutant spectrum included albina, xantha, chlorina, viridis, tigrina, striata, albo-xantha and albo-viridis. The frequency of these mutants varied with treatments. A dose rate of 60 krad was found to be effective as well as efficient for inducing chlorophyll mutations in M 1 and M 2 plants.

The mutagenic effectiveness of 8.5 and 24.5 keV neutrons in Barley

The mutagenic effects of intermediate energy neutrons of 8.5 and 24.5 keV are compared with those of of higher energies (100 and 200 keV and fission neutrons). Due to the low dose-rate of intermediate neutrons and the small space available for irradiation, embryos of resting seeds of Bonus barley were used for the study. The relative biological effectiveness (RBE) of these neutrons was obtained by comparison with the effectiveness of 180 kV X-rays in inducing chlorophyll mutations. The following RBE values of mutagenic effectiveness of neutrons of different energies were found (with 80% confidence intervals); 8.5 keV: 0 (neg.−3.3); 24.5 keV: 21 (12–33); 100 keV: 204 (120–330); 200 keV: 102 (49–190); fision neutrons, ∼ 1MeV: 37 (29–47). However, the difference in RBE between 100 and 200 keV neutrons was not significant in the present case. The low mutagenic effectiveness of low-energy neutrons can be ascribed mainly to the short range of the charged particles. According to the two-hit theory for the induction of chromosomal aberrations by ionizing radiation, this would mean that mutations induced with the high RBE of high-LET radiations are mainly chromosomal aberrations (multi-locus deletions).

Mutagenic Effects on Chlorophyll Mutation Frequencies and Spectrum in Rice

Seeds of the rice variety Co 37 were subjected to gamma rays and EMS alone and in combination with two treatments. The effects of frequency and spectrum were studied. The frequency of mutations estimated as number of mutants per 100 My spikes was as- efficient as the number of mutants per 100 M, plants. Gamma rays proved more potent in inducing chlorophyll mutations than EMS. The combination of treatments induced a higher frequency of chlorophyll mutations than individual treatments showing antagoni stic effect on M, spike and additive effect on M, seedling basis, Regarding the spectrum. of chlorophyll mutations gamma rays induced Albina, Xantha and Striate types, whereas EMS and combination treatments produced Virescent in addition to three types obtained through gamma rays. Albine and Xantha occurred more frequently than other types in all the treatments but the combination treatments produced more Albina than individual treatments.

Mutagenic Effects of Sodium Azide in Rice1

Seeds of rice (Oryza sativa L. spp. japonica) cultivar ‘M5’ were presoaked in distilled water and treated for 2 or 3 hours with 0, 0.12, 0.50, 0.75, 1.0, 1.25, 1.50, and 1.75 mM sodium azide solutions prepared in 0.1 M phosphate buffer (pH 3). After treatment the seeds were rinsed for 1 hour in 15 C tap water. Seeds receiving a 2‐hour azide treatment were redried in a fume hood at room temperature for 24, 48, 72, and 96 hours prior to planting to develop treatment procedures suitable for transporting dry, treated seeds rather than growing seedlings. Although the scope of this study did not permit field evaluation of the redried treated seeds, it was found that seeds treated for 2 hours with I mM azide and redried for 96 hours had more injury than seeds treated for 3 hours with I mM azide without redrying. Thus, a 1 mM azide treatment for 2 hours with redrying may be near the maximum permissible for sufficient field viability of the M1. Seeds with a 3‐hour azide treatment were planted immediately after rinsing. The seedlings were transported by air to the California test site and transplanted for the mutation study.Criteria used to assess the biological effects of azide on rice were germination, seedling height, and seed sterility in the M1 generation, and chlorophyll‐deficient seedlings and viable mutations in the M2 generation.In general, an increase in azide concentration, along with an increase in the post‐treatment redrying period, resulted in a decrease in M1 germination and seedling height. Azide treatment also induced sterility, the highest concentration producing maximum (62.2%) seed sterility. The same treatment induced chlorophyll mutations in 98.5% of the M1 panicle progenies and in 14% of the M2 seedlings. The viridis type of mutation was most frequent.The highest frequency of viable mutations scored in the adult plant stage was 4.64% on an M2 plant basis. All azide concentrations were mutagenic. Short‐culm and dwarf mutant types were more frequent than the early‐flowering mutant types in the azide‐induced mutation spectrum. Azide should prove useful as a mutagen for the genetics and practical breeding of rice.

Mutagenicity of gamma rays and E. M. S. and their combination in Bengalgram (Cicer arietinum L.)

Physical (gamma rays) and chemical (EMS) mutagens and combination of these two (Gamma-+-EMS) reduced the percentage of germination, survival, seedling growth, drymatter production and seed fertility in bengalgram with increase in dose of mutagens. EMS produced greater reduction than gamma rays. Combination treatments showed enhanced effect com- pared to single treatments. Among the varieties CO. I was more sensitive than G 62404. A linear dependence for all the characters studied on dosage was evident in both the varieties. The same trend was noted in the induction of chlorophyll mutations. Chemical mutagen was more potent in inducing chlorophyll mutations.

Studies of induced chlorophyll mutations in Lathyrus sativus L.

Dry and dormant seeds (10% moisture) of Lathyrus sativus L. var. P10, P24, P288, P293, P585 and LC76 were irradiated with 10, 20, 30, 40 and 50 kR gamma rays. Both control and irradiated seeds of variety P288 was treated with 0.2 MES for six hours. Albino, Xantha, Albo-Xantha, Xanthalba, Albo-viridis, Virescence, Chlorina, Albescence, Tigrina and Maculata were the general types of chlorohyll mutations observed in all the six varieties at M2 generations.The spectrum of chlorophyll mutations was found to be dependent on the nature of genotypes. This was further noted to have linear relationship with doses. Maximum chlorohyll mutations were observed in LC76 which was proved to be more sensitive than rest of other varieties.High frequency of mutations was recorded in P288 as a result of differential and combined treatment of 0.2% MES which was seen to be as effective as 20 kR gamma rays.

Organophosphate induced chlorophyll mutations in Hordeum vulgare

Ten Organophosphorus (OP) insecticides are tested for their genetic toxicology in the Hordeum vulgare system. Of these, 8 OPs induced chlorophyll mutations of which 6 are new discoveries.

LSD induced chlorophyll mutations in barley

In earlier reports, LSD was found to induce extensive chromosomal aberrations in barley. With the same dose it is now observed that LSD is potent in the induction of chlorophyll mutations. This may be the first evidence that the drug induces gene mutations in plants.

Modification of mutagenic efficiency of ethyl methanesulfonate with ethidium bromide, iodoacetamide and sodium fluoride in barley

In order to modify the cellular sieves through which a pre-mutational lesion must pass before it can be realized as a phenotypic change, barley seeds were treated with ethyl methanesulfonate (EMS) in combination with non-toxic concentrations of ethidium bromide (EB), iodoacetamide (IA) and sodium fluoride (NaF). Treatments with EMS, EB, IA and NaF alone served as controls. Pre- and post-treatments with the modifying chemicals were also investigated. Seeds treated with EMS and three different modifiers served as the various controls. The parameters considered were germination, seedling injury, plants with chlorophyll chimeras, mitotic and meiotic aberrations, pollen sterility and seed set in M 1 and the frequency of chlorophyll mutations in M 2. None of the modifiers at the concentrations used, induced chlorophyll mutations in M 2. However, the presence of EB, IA and NaF at the same concentration during treatments with EMS, significantly enhanced the mutagenic effect of the latter. Pre- and post-treatment with IA also significantly enhanced the mutation frequency in M 2 seedlings. Pre- and post-treatments of EB and NaF did not bring about significant alterations in the mutation rates.

Effects of sodium acetate and sodium chloride on EMS-induced chlorophyll mutations in barley

Sodium acetate solutions to which sodium chloride was added, and acetate or chloride alone have been used as pre-, simultaneous, and post-treatment of dry and pre-soaked seeds of barley to study their effect on the types and frequencies of ethyl methanesulphonate (EMS)-induced chlorophyll mutations in spring barley, variety Elsa, and winter barley, varieties 436 35 and Ager. Application of acetate/chloride on dry seeds before or simultaneously with EMS both resulted in the frequency of chimeral plants with chlorophyll-deficient sectors in M 1 and chlorophyll mutants in M 2 approximately being halved as compared with the controls (EMS treatment alone). An opposite effect was observed after simultaneous treatment with acetate/ chloride and EMS (pH 4.5 and pH 7.0) and application of acetate/chloride after EMS treatment of pre-soaked seeds. In this case the mutagen sensitivity, i.e. the frequency of chimeral plants with induced chlorophyll-deficient sectors in M 1 and of chlorophyll mutants in M 2, was approximately doubled as compared with the control. Separate application of both acetate or chloride as a simultaneous treatment with EMS resulted also in an increase in the chlorophyll mutation frequency as compared with EMS treatment alone. Based on these results some aspects of the acetate/chloride effect are briefly discussed.

Studies on the cytogenetic and genetic effects of fluoride on barley. II. The effects of treatments of seedling coleoptiles with sodium fluoride.

Treatment of seedling coleoptiles (truncated and non-truncated) with either sodium fluoride alone or in combination with dimethyl sulfoxide did not inhibit growth. Cytological analyses indicated that these treatments produced chromosome abnormalities, including bridges with or without fragments, univalents, fragments and micronuclei. The production of a significantly higher percentage of chromosomal aberrations by simultaneous treatment with a low concentration of sodium fluoride and dimethyl sulfoxide over that produced by treatment with low concentration of sodium fluoride alone indicates that dimethyl sulfoxide can enhance the uptake of the mutagen and thereby increase the mutagenic effect.Tests were conducted to determine whether the fluoride treatments were capable of inducing chlorophyll mutations. Plants whose coleoptiles had been treated at the seedling stage were self-fertilized and the S1seeds obtained used to produce plants which were self-fertilized. The S2seedlings obtained from these latter self-fertilizations were screened for chlorophyll mutations. None was observed.

Mutation induction by EMS in autotetraploid barley

The mutagenic efficiency of radiations (fast neutron, gamma- and X-rays) and chemical mutagen (EMS) was compared on induced autotetraploid barley. It was found that only EMS was effective in inducing chlorophyll mutations in the M2 generation. The theoretical basis of this finding is discussed.

Radiation genetics in wheat—IX: Differences in effects of gamma-rays and 14 MeV, fission and fast neutrons from Po-Be

Dry seeds of Triticum monococcum were exposed to Cs 137 γ-rays or to monoenergetic 14 MeV neutrons obtained from the T ( d, n) reaction in a neutron generator. For both radiations the higher the dose, the more delayed the seedling growth and the more reduced both survival rate and seed fertility. However the frequency of chlorophyll mutations increased with increase in dose. These results are in good accord with those obtained in our earlier work with 14 MeV neutrons provided by the Oak Ridge National Laboratory, U.S.A. (ORNL). (11,12) From the data from both experiments the RBE of 14 MeV neutrons vs. γ- and X-rays was found to be about 10 for germination rate, 13 ~ 15 for seedling growth and about 15 for seed fertility and chlorophyll mutation rate. The fission neutron (average 1–5 MeV) irradiations of dry seeds at three ploidy levels were conducted in the ORNL reactor and parallel γ-irradiations from Co 60 were also carried out at ORNL. In general, T. monococcum ( n=7) was the most sensitive to fission neutrons and γ-rays. There was, as expected, no significant difference between T. durum ( n=14) and T. vulgare ( n=21). The higher the dose of fission neutrons and γ-rays, the more delayed were germination and seedling growth, and the more reduced was seed fertility. As expected, the higher the ploidy, the lower the frequency of chlorophyll mutations. For seedling height the RBE value of fission neutrons to that of γ-rays was found to be 10 ~ 15 in the three ploidy series. For seed fertility the RBE was about 10 for diploids and about 50 for tetra- and hexaploids and for chlorophyll mutations an RBE of 25 or more was determined for di- and tetraploids. In general, the RBE value was higher for the characters observed at maturity than in earlier stages, especially in polyploids. Fast neutrons of mean energy 4·7 MeV (16) were obtained from a Po-Be source. Doses of 75 ~ 125 rad were not markedly effective in inhibiting seed germination and seedling growth or decreasing survival and seed fertility in T. monococcum, but were effective in inducing chlorophyll mutations. It is concluded that these neutrons are more effective than 14 MeV and fission neutrons, and that the RBE value for chlorophyll mutation frequency is about 40 against γ-rays.

Types and frequencies of chlorophyll mutations in Durum wheat induced by radiations and chemicals

The comparative effects of X-rays, thermal neutrons, fast neutrons, diethyl sulphate and ethyl methanesulphonate were studied, using dry seeds of the cultivated varieties Aziziah, Cappelli and Russello of durum wheat. Aziziah was found as the most radiation sensitive variety; no statistically significant difference in radiation sensitivity between Cappelli and Russello was ascertained. Excluding the results of two chemical treatments, the three varieties studied did not differ from each other as to frequency and spectrum of chlorophyll mutations in the second generation (M 2). At comparable survival rates, mutation and mutant frequencies in M 2 were two to several times higher after neutron irradiation than after X-rays. In the radiation series, maximum rates of chlorophyll mutations were found in treatments producing a 50 to 80 per cent reduction in the mean number of M 2 seedlings per spike; in the most efficient chemical treatments, the reduction in seedling number per spike comprised between 33 and 42 per cent. Chemically induced spectra of chlorophyll mutations were quite distinct from those resulting from irradiation; they were characterized by a reduced frequency of albina and a greater frequency of xantha and viridis. If the distribution of albina, xantha, chlorina and tigrina was considered in relation to seedling number per spike, they showed a clear accumulation in lower spike fertility classes in the radiation series. In the chemical mutagen series, albina, xantha and chlorina occurred frequently in spike classes with good fertility, whilst tigrina typically accumulated in spikes with clearly reduced fertility. Evidence was found that, in chemically treated materials, the mean size of mutated sectors in M 1 spikes is much smaller than following irradiation. It was estimated that, in several M 1 spikes from chemical treatments, four or possibly more initial cells were to be made responsible for the organization of a spike. Evidence is presented that the deficit of recessives shown in M 3 by individuals heterozygous for radiation-induced chlorophyll mutations can be clearly reduced in the transition from M 3 to M 4. Five out of 1024 induced chlorophyll mutations were found to behave as dominant or semidominant.