R.A. Nilan

Induction and selection of specific gene mutations in hordeum and pisum

Barley (Hordeum vulgare L.) and pea (Pisum sativum L.) seeds were treated with the mutagen sodium azide. The second generation (M2) seeds or seedlings were screened for physiological (nitrate reductase-deficient and chlorate-resistant) mutants and presumed single locus (waxy endosperm and vine) mutations. Nitrate reductase-deficient mutants occured at an overall frequency of 13.1 per 10,000 seedlings in barley and 3.5 per 10,000 seedlings in peas. Chlorate-resistant mutants occurred at a frequency of 6 per 10,000 seedlings in barley. Waxy endosperm mutants occurred at a frequency of 2.7 per 10,000 seeds and vine (gigas) mutants occured at a frequency of 1.0 per 10,000 seedlings in barley. These data demonstrate that specific gene or function mutations occur with high frequency in azide-mutagenized barley and peas and indicate that similar loss of function mutants could be selected for in higher plants with even relatively laborious screening techniques.

THE EFFECT OF ETHYL METHANESULFONATE ON THE GROWTH RESPONSE, CHROMOSOME STRUCTURE AND MUTATION RATE IN BARLEY

Abstract Effects of ethyl methanesulfonate (EMS) and X-rays were compared by several biological measures in barley. The criteria studied included rate of growth, survival and fertility of M1 plants, frequencies of chromosomal aberrations in mitosis of seedlings and meiosis of M1 spikes, and the frequencies of chlorophyll deficient mutations among M2 seedling progeny. The doses of EMS and X-ray treatments chosen reduced the rate of seedling growth and survival to a comparable degree. The reduction of fertility was directly related to the frequency of chromosome aberrations and mutations after X-ray treatment. After EMS treatment, the decrease of fertility was pronounced; however, there was little effect on seedling growth and survival. Also, the frequency of chromosome aberrations observed in EMS treatments was negligible, whereas a high rate of chlorophyll deficient mutations was induced in both M1 plants and M2 progenies. Preliminary results indicated that the efficiency of EMS treatments can be further increased when the treatment solutions are buffered and the delayed action of the mutagen is prevented. Physiological damage, e.g., reduced seedling growth and survival, increased with concentration of hydrolysis products in the treatment solutions, except when buffers were added. The damage caused by drying seeds after EMS treatment was reduced considerably by soaking the treated seeds in distilled water before drying. This delayed effect may be due to active agent and/or by-products retained in the tissue after the defined treatment time.

POST-IRRADIATION OXYGEN SENSITIVITY OF BARLEY SEEDS VARYING SLIGHTLY IN WATER CONTENT.

Abstract Himalaya barley seeds ranging in water content from 9 to 12 percent and differing by only 0·2–0·3 percent were irradiated in a vacuum with 25 krad of 60Co γ-rays. After irradiation they were immediately (within 1 min) hydrated in either oxygen- or nitrogen-bubbled water at 0 °C for a period of 16 hr. Results, as reported in terms of seedling injury, indicate that there is a very critical region in which a small change in water content greatly alters the oxygen-dependent damage. In a region of only 0·3 percent water content change, i.e. between 10·7 and 11·0 percent, the response to oxygen dropped by a factor of more than 2·5. The oxygen-independent damage was not influenced by a change in seed water content in the 9–12 percent region. Based on the hypothesis that oxygen and radiation-induced free radicals interact to produce at least a part of the biological damage, it would appear that mobility, and hence, interactions of oxygen-reactive radicals have rather specific water requirements.

Mutagenic and chromosome-breaking effects of azide in barley and human leukocytes ☆

Azide (10-3 M, solution buffered at pH 3) is more effective in inducing mutations in embryonic shoots of seeds germinated between 8 and 16 h than in non-germinated seeds and in seeds germinated between 0 and 8 h and 16 to 28 h. This peak of chlorophyll-deficient seedling mutation frequency coincides with maximum frequencies of seeding lethals and DNA replication in the cells of the embryonic shoot. The mutation data suggest azide may only act on replicating DNA. Azide induced no chromosome-aberration frequencies significantly above controls in (1) embryonic shoots of barley seeds germinated for 8--12 h, (2) microspores of barley and (3) human leukocytes. It appears to be a point-mutation mutagen.

Differential effect of sodium azide on the frequency of radiation-induced chromosome aberrations vs. the frequency of radiation-induced chlorophyll mutations in Hordeum vulgare

A considerable synergistic increase of the frequency of cells carrying chromosome aberrations was observed in seeds of Hordeum vulgare , variety Himalaya, irradiated dry with gamma rays and hydrated in sodium azide solutions buffered at pH 3, 7 and 11. This effect, which was most evident at pH 3, was detected throughout development up to the formation of the male gametophyte. No synergistic increase of the frequency of radiation-induced chlorophyll-deficient mutants was observed. This differential effect of the sodium azide post-irradiation treatment on the frequency of chromosome aberrations vs. the frequency of mutations is attributed to the formation of two kinds of lesions in the genetic material of barley following exposure to gamma rays. One kind of lesion, similar to those leading to point mutations in procaryotes, is reparable at low energy potentials and, thus, its frequency is not affected by the sodium azide treatment. The other kind of lesion is associated with chromosome breaks and its repair is affected by the presence of the enzymatic inhibitor.

The effect of sodium azide on cell processes in the embryonic barley shoot

Sodium azide has been utilized recently both as an agent for the study of repair of radiation-induced chromosome damage, and as a mutagen in barley caryopses (seeds). However, the effect of this agent on the cell cycle and optimum time of treatment during the cell cycle in these studies is not known. To better understand the effects of sodium azide on the embryonic barley shoot cells, a detailed study of the effect of azide on the cell cycle was conducted. Himalaya barley seeds were treated for 2 hr with 10−4, 5 × 10−4, and 10−3M oxygenated sodium azide solutions at pH 3. The principal effect on the cell cycle due to sodium azide treatment was a delay in the initiation of metabolism following germination. This resulted in a uniform delay in the following parameters: mitotic activity, seedling growth, and ATP and DNA syntheses. This delay was interpreted as being due to an ATP deficiency which when alleviated allows the cells to progress normally through mitosis. Chromosome damage caused by sodium azide was not reflected in the seedling heights as the reduction in height was due entirely to mitotic delay. No variation occurred in the progression of cells through mitosis between various regions of the shoot within the first 29 hr of germination.

THE ROLE OF INDUCED MUTATION IN SUPPLEMENTING NATURAL GENETIC VARIABILITY

Plant breeding or improvement involves the same basic ingredients and processes as plant evolution; namely, genetic variability and selection. Genetic variability consists of genes and gene forms or alleles produced by mutations and recombination. The latter is produced in turn by meiotic or mitotic crossing over and by independent assortment. The resultant genotypes are selected either by natural processes or the plant breeder’s artificial techniques. From the above it is clear that the truly basic ingredient of plant breeding and, indeed, plant evolution, is mutation. This can be broadly described as all genetic alterations, ranging from single base substitutions within the DNA comprising the gene to gross changes in chromosome number and structure, which cannot be accounted for by recombination. When only a relatively few of these genetic changes occur, as through spontaneous mutation, there can be only a limited number of new recombinations and, hence, only slow evolution. To achieve the rapid evolution needed by plant breeders for satisfying the rapidly changing needs for food and fiber, more rapid generation of new genetic forms is required. Such requirements for large amounts of new genetic variability can only be met with the aid of induced mutations as supplements to natural genetic resources. Geneticists know of no other way to “manufacture” new gene forms or alleles other than by mutagenic agents. Induced mutations should become increasingly important sources of genetic variability for plant improvement programs because (1) sources of natural genetic variability for some crop plants are at various stages of depletion and (2) induced mutations represent a new, nearly untapped reserve of genetic variability. This paper will acquaint the reader with modern induced-mutation technology, its successes and problems, and evidence that induced mutations can aid in compensating for lost natural diversity and in alleviating genetic vulnerability in plants.

Effect of gamma radiation on gibberellic acid solutions and gibberellin-like substances in barley seedlings*

Aqueous solutions of 10−6 m gibberellic acid, irradiated with relatively low doses of gamma rays, failed to induce α-amylase activity in embryoless barley caryopses. The relationship between irradiation dose and gibberellic acid inactivation is an exponential one. Presence of the free hydroxyl radical scavengers, potassium iodide and p-aminobenzoic acid, protects the biological activity of gibberellic acid as it is measured by the α-amlyase bioassay. These results suggest that gibberellic acid in aqueous solutions is inactivated by gamma rays by an indirect action, probably through reactions with free radicals from the radiolysis of water. A reduction in the absorbance peak at 1750 cm−1, corresponding to the γ-lactone ring, follows exposure of gibberellic acid to gamma rays. Irradiation of young barley seedlings does not affect their content in gibberellin-like substances, but in seedlings from irradiated caryopses the concentration of the gibberellinlike substances is reduced.

The effect of ethylene and ionizing radiation on Saintpaulia peroxidase activity

Abstract Ethylene gas and X rays, alone and in combination, were applied to petioles of Saintpaulia ionantha Wendl. to test their effect on peroxidase activity. Both agents increased peroxidase activity, although ethylene was the most effective. X ray treatment of petioles at five different peroxidase levels showed that as the initial activity of peroxidase increased the effect of a given dose of radiation decreased. Also, high doses, near the lethal limit for the species, were proportionately less effective in inducing peroxidase activity than were lower doses. Polyacrylamide disc electrophoresis showed no change in isozyme patterns with increases in peroxidase activity.

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON RADIATION-INDUCED DAMAGE IN BARLEY

Abstract Resting seeds (14% moisture content) of barley (Hordeum vulgare var. Himalaya) were gamma irradiated and then hydrated in buffer solutions at different pHs for 12 hr at 20°C. The response of the irradiated seeds to hydrogen ion concentration of the hydration solution was measured by several biological criteria. A modification of the radiation-induced damage as measured by M1 seedling growth injury, lethality and mitotic chromosome aberrations was found. The general trend was a decrease in radiation-induced damage with an increase in the pH of the hydration solution. In contrast, the frequency of radiation-induced M2 seedling mutations and M1 plant sterility were influenced but little. Mutagenic efficiency, i.e. the ratio of mutations to amount of “damage” e.g. seedling injury, mitotic chromosome aberrations and lethality, induced by gamma radiation, was greater at basic pHs than at acidic pHs. It was hypothesized that hydrogen ion concentration might influence the radiation-induced chromosome aberration frequency by either (a) modifying the properties of radiation-induced radicals responsible for the induction of chromosome breaks, or (b) by altering the activity of enzymes concerned with the development and/or subsequent behaviour of chromosome breaks. The latter mechanism is favoured.