"These experiments," he writes, "require a source of X or gamma rays. In general the irradiation facilities of universities and Government agencies cannot be diverted for amateur use because they are fully occupied by research programs. Medical and industrial radiation sources, however, seem to be sufficiently numerous and widespread so that occasional access to them can be had if the experimenter has prepared the specimens with an eye to conserving the time of X-ray technicians and if he can demonstrate the value of his objectives. It was with these considerations in mind that seeds were selected for the experiments that follow. Seeds-actually dormant plant embryos-are attractive experimental materials in a number of respects.

"A seed is in a state of suspended animation; when provided with an environment that encourages germination, it becomes a typical living system. A seed is a complex, multicellular organism that is obedient to the laws of heredity. This means that all individual plants that grow from seeds of a 'pure line,' particularly those of a self-pollinating species, are almost identical genetically. They contain all the types of cells of the future plant telescoped into a small volume. Since these cells are in a resting state the experimenter can make identical observations over an extended period. In general seeds tolerate extremely harsh treatment; they can therefore provide biological assays of treatments severe enough to kill most other living organisms, such as extremes of radiation, harsh chemicals and high or low temperatures and pressures. Unlike mature plants, seeds can be manipulated conveniently and a large number can be irradiated simultaneously by a compact source. Of most interest, the experimenter can measure the effects of radiation by counting the number of seeds that germinate, measuring the rate of seedling growth, counting the number of plants that survive to maturity and observing such characteristics as fertility (in terms of seed set), chromosomal aberrations in cells, pollen abnormalities and mutations in the second and later generations.

"The seeds of different plants vary greatly in their sensitivity to radiation. Cabbage, for example, can withstand 200 times more ionizing radiation than an onion or a lily. The reasons for such variations are of deep biological significance and their discovery by experimental methods can be a challenging and deeply satisfying experience. In an environment of increasing radiation it is important to learn the effects of radiation on seeds and to find methods of utilizing and modifying their consequences.

"The selection of seeds is the first step in planning a radiation experiment. Specimens should be genetically uniform; otherwise the effects induced by radiation might be confused with existing genetic differences. Radiation botanists customarily use seeds of a self-pollinating species that can be traced to u single genetically pure ancestral plant. Certified agricultural seeds or those of varieties that can be procured from a reputable firm are adequate for the suggested experiments that follow. Seeds sold by the pound are less costly than those that are sold in small, colorful packages. The selection of seed species for radiation experiments is limited principally by the energy of the radiation source. If a high-energy machine is available, almost any species can be used, but if the source is of low voltage or if exposures must be kept short for economy or other reasons, sensitive seeds should be chosen. The accompanying table [Figure 1] lists the relative radiation sensitivities of some common plants. The indicated doses are approximate, because seedling response varies with the age, variety and moisture content of seeds, the kind of radiation source, filters, if any, growing conditions and other factors.

"Having selected one or more species, first sort the seeds by hand and discard any that are broken, discolored, outsize or otherwise abnormal. Larger seeds of a given species in general produce more growth than smaller seeds. Mixed sizes can lead to subsequent error when the effects of radiation are assessed. Time spent in hand-picking seeds will be rewarded by the increased validity of the results.

"The number of seeds used for each experiment is determined by the size of the seeds, the medium in which they are grown and the purpose of the experiment. As a rule, variations in both seeds and X-ray machines are large enough to obscure radiation effects in a sample of 10 seeds or fewer. Fifty seeds per treatment is a reasonable number, although 100 is the minimum for mutation studies. An unirradiated group of seeds must always be grown as a reference, or control.' The control should always consist of the same number of seeds as the treated lot, selected with the same care and grown with the same amount of water, nourishment and cultivation.

"After the seeds have been counted, packaged and labeled for treatment, each group should be spread on the growing medium in a single disk-shaped layer that corresponds to the size of the X-ray beam. The intensity of the beam may not be uniform. If the intensity varies from area to area within the beam, either choose a small region of uniform intensity for the exposure, place the seeds on a small motor-driven turntable for treatment or interrupt each treatment several times during irradiation and shift the position of the seeds. (An experiment for determining the uniformity of the X-ray beam will be described later.)

"If some factor other than radiation is to be varied, such as moisture or the gaseous environment, each sample should be sealed in a material such as plastic kitchen wrapping, heavy polyethylene or glass to maintain the desired environment. When preparing such packages, remember to keep the seeds in a single layer for irradiation unless the source of radiation is highly energetic (one million electron volts or more) to assure penetration to a depth of several centimeters.

"In these experiments the amount of radiation to which the seeds are exposed will be specified in kiloroentgens, or thousands of roentgens. The roentgen is the amount of radiant energy required to dislodge electrons from approximately two billion molecules in one cubic centimeter of air at a temperature of t32 degrees Fahrenheit and a pressure of 760 millimeters of mercury. Thus one roentgen generates two billion ions in one cubic centimeter of air. Observe that time is not a factor of the unit. A low-energy X-ray source can create two billion ions just as effectively as a high-energy source if the machine operates long enough. Few medical and industrial X-ray machines are calibrated in roentgen units; exposures are based on quantities such as voltage and time.

"The output of X-ray machines varies with many factors, including the kind of tube and its age, the applied voltage and current and the distance from the tube to the specimen. Most X-ray technicians have 'R' meters, however, and can measure the energy of the beam in terms of roentgens at a desired distance from the tube. (Recently the 'rad' has become a popular unit of radiation, principally because of its convenience in making computations. The rad corresponds to the absorption of 100 ergs of energy by each gram of tissue, whereas the roentgen corresponds to the absorption of 93 ergs. To convert roentgens to rads multiply by .93.)

"Most seeds require only water and air for germination at room temperature (60 to 70 degrees F.). Those of wheat and beans have sufficient stored food to support growth for two to three weeks, but smaller seeds may require prompt nourishment when they sprout. Seeds can be grown in shallow boxes of sand, peat, perlite, soil or mixtures of these materials. The growing medium should be kept moist but not soggy and should be fertilized every three or four days with a soluble plant food; commercial preparations that are recommended by seed dealers will do.

"Seeds with abundant stored food can be grown to sufficient size for seedling studies in paper towels or blotters that are rolled and placed in shallow containers of water [Figure 2]. The correct temperature must be maintained, measured in the soil. Seeds normally sown in late spring or early summer, such as corn, cotton and soybean, require a soil temperature of about 70 degrees, whereas those that sprout in the fall or early spring, such as spinach, onions and most grasses and clovers, thrive at 60 degrees or less. Proper germinating conditions for most field and garden seeds are listed in the publication Rules and Regulations under the Federal Seed Act, which can be obtained from the U.S. Department of Agriculture.

"If seeds with little stored food are used, the blotters should be made narrow, perhaps an inch wide, so that the roots will soon contact the liquid in the dish. Remember that roots need oxygen. Oxygen escapes from a liquid in a few hours, but it can be replaced by changing the nutrient solution daily. Always place new solutions in large bottles with plenty of air space and before use shake them vigorously for several minutes so that they will absorb the maximum amount of oxygen. When the first leaves appear on the plants, rotate the roll of blotters and shift the dishes in a regular daily pattern to minimize the effects of unequal light and temperature.

"You will quickly observe that mere sprouting, or germination, is a poor criterion of radiation damage. Virtually all seeds germinate, even those subjected to lethal doses of radiation. They do so because most germination results from the elongation of cells already present in the embryo; it is a further increase in size that depends on cell division, which can be inhibited by radiation. For this reason radiation damage can be assessed reliably only by measuring the growth of seedlings. A reasonably accurate indication of radiation damage in the case of 'monocots' such as wheat and onion, which send up a single first leaf, can be obtained by measuring the height of all irradiated seedlings when the leaf of the control group is fully extended.

"Damage is more difficult to measure in the case of 'dicots' such as spinach and beans, which raise two seed leaves by elongation before growth by cell division begins. In the case of these plants the distance between the original location of the seed and the leaves remains relatively constant through a wide range of radiation dosage, and growth above the leaves may amount to only a few millimeters in three weeks. In such plants the dry weight of the tops is a reliable measure of growth. The optimum time for harvesting is determined by observing the growth of the controls. It will vary from about 10 days for wheat or beans, which grow four to six inches during this interval, to about four weeks for clovers, which will have reached a height of scarcely two inches.

"A graph made by plotting seedling growth against time takes the form of an S-shaped curve, and the time to harvest is when the curve of the control begins to flatten out. Clip the seedlings at 'ground level,' chop up the plants from a given irradiation treatment, place them in a drying dish of known weight and label them. Dry the material in an oven at 150 degrees for 24 hours and remove to a desiccator for cooling. Weigh each cooled dish and subtract the weight of the dish from the total weight. The dry weight of the irradiated seedlings divided by the dry weight of the controls and multiplied by 100 equals the percentage of growth of the treated group in relation to that of the control group. Record this percentage in a notebook for each group.

"These techniques are similar to but simpler than those employed by radiation biologists. Gross differences of behavior must be expected even among control groups when single measurements are made of small numbers of seedlings. In some experiments plants from heavily irradiated seeds may appear to have grown more than those that received a lower dose, or a lower dose may seem to have promoted better growth than a control. Such observations can usually be traced to faulty technique: miscounting seeds, mislabeling treatments, differences in light or temperature, mistakes in watering and so on. Random variation, or 'error,' is always greater in small samples than in large samples. To learn how much natural variation to anticipate make an experimental run with six to 10 groups of untreated controls. To detect subtle changes induced by ionizing radiation you must use large numbers of seeds and, in particular, repeat experiments at least four times. Only then can statistical tests be applied with confidence.

"For a simple introductory experiment select two species, one known to be sensitive, such as onion or rye, and one that is resistant, such as mustard or cabbage. After hand-picking, divide the seeds into four groups of 50 or 100 seeds per group, depending on how many seeds the X-ray machine will accommodate when specimens are spread as a single layer. Package each group separately and label for species and radiation treatment. Irradiate the seeds of both species simultaneously, reserving one group of each for controls. The treated groups should receive doses of one kiloroentgen, 10 kr and 100 kr respectively. Measure the dry weight of all seedlings to compare the response of the two species to irradiation. Differences of response have been ascribed to the genetic structure of the organisms, but this does not explain the responsible mechanism. Some recent evidence indicates that 'target sizes' and differences in the geometry of cells and nuclei are involved, as well as 'sensitive sites' in the cells, but the question of why some plants are more sensitive to radiation than others remains open.

"A second experiment, for determining the uniformity of the X-ray beam, can be run simultaneously. Cut a disk of cardboard equal in diameter to the beam of the X ray, draw a circle in the center Of the disk equal in diameter to the radius of the disk and divide the area outside the circle into four equal parts. Select seeds of a plant of fairly high sensitivity, divide the seeds into five equal groups and place them as single layers in the five inscribed areas of the cardboard.

"One axis of the pattern should be placed perpendicularly to the long axis of the X-ray tube and a note made of its position with respect to the tube. Label each group of seeds and administer a significant dose of radiation as listed in the table. Grow the groups separately and compare the dry weight of the seedlings in each group with that of a single unirradiated control group. Finally, make a note of the percentages of relative growth in each area of the cardboard disk. X-ray beams characteristically take the form of nonuniform cones of radiation of highest intensity on the anticathode side of the X-ray tube. If the beam is producing growth differences of more than 25 per cent in different areas, special steps will have to be taken to compensate for the disparity during subsequent experiments. Either shift the seeds periodically during irradiation or rotate them on a small turntable such as those used for advertising displays.

"Resistance to radiation damage appears to vary not only from plant to plant but also from day to day in a plant that is germinating. To observe this difference start two lots of labeled seeds. On the following day start two more groups and on the third day start yet another pair. On the fourth day irradiate one lot of each pair with one-fifth of a significant dose. At the same time plant another two lots, having previously irradiated one of them. Grow and harvest the seedlings and measure their dry weights. Plot, as a graph, the days of germination prior to irradiation (zero to three) against the dry weight of each treated group as a percentage of its control; observe that the sensitivity of seeds to radiation damage increases as they become biologically active.

"Moisture also strongly influences sensitivity to damage. Store groups of seeds for at least a month in a series of controlled relative humidities. (Do not use seeds of rye, corn, barley, wheat, millet or other grasses for this experiment.) Controlled atmospheres can be set up in wide-mouthed glass jars equipped with close-fitting lids and inner devices for supporting the seeds above a fluid. Saturated aqueous solutions of the following salts will produce the indicated relative humidities: zinc peroxide, 10 per cent relative humidity; lithium chloride, 15 per cent; calcium chloride, 32 per cent; sodium bisulfate, 52 per cent; sodium chloride, 76 per cent, and potassium nitrate, 94 per cent. Care must be taken to maintain constant room temperature or condensation will moisten the seeds and destroy the experiment.

"At the end of the storage period quickly divide the seeds from each jar into four equal groups and seal immediately in plastic kitchen wrapping. (Do not take time to count the seeds at this juncture as they will tend to approach the humidity of the room.) Label each package for relative humidity and radiation dose. Retain one lot from each jar as a control. Irradiate the remaining three groups at the significant dose, twice the significant dose and five times the significant dose respectively. Grow and measure by the routine procedure, counting the number of seeds (not seedlings) as each group is harvested. Compute the growth of each group in terms of milligrams of dry weight per seed sown. Plot the results with the percentage of relative humidity as the horizontal axis and the growth as the vertical axis. Observe that the sensitivity of seeds to ionizing radiation varies with the humidity at which they are stored before and during irradiation. For each species there is a different optimum humidity, both for unirradiated growth and for resistance to radiation.

"Sensitivity appears to be similarly influenced by exposure to gases. To observe the effect place groups of selected seeds in plastic containers, such as the freezer bags normally used for preserving foods. Flush some lots for about two minutes with oxygen and an equal number with nitrogen or helium and seal the containers promptly. After allowing the gases to diffuse into the embryos for 24 hours or more, remove the seeds and immediately irradiate each group with a significant dose. Compare the seedling growth of each gas-treated group with its control and compare the gas-treated groups with one another. It can be shown by this experiment that radiation damage is caused in part by the reaction of induced radicals with oxygen to form active substances that induce hereditary changes in plants; seeds stored in air or oxygen are more severely damaged by radiation than those stored in inert gases.

"The experiments discussed so far were designed to show radiation damage as measured by seedling growth. Such damage is partly genetic and partly physiological. Only the genetic changes are transmitted to offspring, and virtually all genetic changes, or mutations, become visible only after a lapse of one generation or more. Any of the above experiments can be extended to an investigation of mutation with the addition of a few steps to the procedure.

"After irradiation sow the treated seeds and controls in a greenhouse or garden plot where they can grow to maturity. Take such steps as are necessary to encourage self-pollination. Depending on the species, this may require bagging each plant or the head of each, manipulating the heads at pollination time, actually transferring pollen from the anther to the stigma (as in the case of corn) or simply isolating the experimental plants from other varieties with which they might cross. Such self-pollination tends to encourage the concentration of recessive mutant genes in the plant and thereby hasten or increase the expression of altered characteristics. In some species inbreeding reduces the vigor of the plant and discloses hidden genes that are also expressed in altered characteristics such as changes in the shape, size or color of leaves or fruit. For this reason an adequate number of controls must be used for distinguishing between altered characteristics induced by radiation and those that can be traced to natural recessive genes.

"Harvest seeds from each mature plant and label by treatment and plant number. These seeds are called the R₂ generation; they were produced on R₁ plants and will in turn produce R₂ plants and R₃ seeds. Sow as many seeds as growing space permits from each R₁ plant when the next growing season arrives.

"Arrange the seeds from one plant in a single row, called a progeny row; if seeds from several plants or treatments are bulked, the mutation study becomes a needle-in-the-haystack puzzle. Examine the R₂ plants for potential mutants at intervals during the growing season.

Look for differences in height and vigor, color and shape of leaves and flowers, date of maturity and so on. Keep careful records. Harvest seeds from as many R₂ plants as growing space permits and repeat the growing cycle the following year. Keep seeds from each plant in a clearly labeled envelope. The plants from these seeds will enable you to verify suspected mutants. Compare original radiation treatments with controls for the percentage of R₂ rows that contain mutants.

"To make a more detailed experiment on the genetic effects of ionizing radiation procure young cuttings or seedlings of a plant that produces showy flowers, such as snapdragon or tradescantia. With the help of a botanist or horticulturist select a variety 'heterozygous' for flower color, that is, one in which a different gene determining color is derived from each parent plant. Before any flower buds become visible irradiate the whole plants, with the exception of the roots, with one or more doses of from 100 to 1,000 roentgens. (Shield the roots with lead if possible.) Keep an unirradiated control group.

"Plant and cultivate, and as each flower reaches maximum size count the number of petal spots of altered color. Each spot so altered represents a single mutation that the geneticist would describe as a change from Aa to aa, the upper-case letter standing for a dominant gene and the lower-case letter for a recessive gene. If the plants were AA to start with, a change to Aa might occur but it would not be expressed by a change in color of the petal spots because the color is determined by the presence or absence of the dominant gene; an Aa would look like the original AA. If the experiment started with petal color determined by aa, then a change to Aa called a 'dominant' mutation, would be expressed in altered flower color, but genetic changes in this direction almost never occur.

"Examine each spot at low magnification and count the number of mutated cells in the spot. You can now deduce how many cell generations ago the mutation occurred: a two-cell spot developed one cell generation ago; four cells indicate that mutation occurred two generations ago; eight cells, three generations, and so on. The larger the spot, the smaller the bud at the time of irradiation-the ultimate being a flower of uniform but changed color, indicating that the mutation occurred when the flower was a single cell.

"For additional data about these and other experiments with irradiated seeds I recommend the booklet Experiments with Radiation on Seeds, Number 2. This reference is available from the U.S. Atomic Energy Commission, Division of Technical Information Extension, Educational Materials Section, P.O. Box 62, Oak Ridge, Tenn."

Bibliography

EXPERIMENTS WITH RADIATION ON SEEDS. Thomas S. Osborne. Division of Technical Information, U.S. Atomic Energy Commission, 1963.

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