Gibberellic acid is now commercially available. It should interest amateurs because it is inexpensive, produces spectacular effects and offers an unusual opportunity for original experiments.. Among those who have worked with it are Robert Lawrence and Henry Soloway, who are students in the College of Medicine of the State University of New York.

"A speck of gibberellic acid smaller than a grain of sugar," they write, "has turned our window box into an Alice-in-Wonderland jungle. The compound causes most plants to grow at record speed, flower in half the usual time and bear fruit that will win first prize in any county fair. In view of these effects and their implications it is not surprising that gibberellic acid is now bringing many strange bedfellows together: the florist seeking to produce larger flowers, the farmer trying to double the production of his land and the cancer researcher who hopes in some manner to find clues to the dynamics and pathology of growth.

"The amateur who experiments with gibberellic acid is likely to reap satisfying rewards because the field is still wide open. Most of the research now under way is centered on crop plants and flowers cultivated by commercial greenhouses. Amateurs can avoid duplicating these experiments by selecting less common plants. For example, to our knowledge no work is being done with molds, mushrooms, mosses, ferns or fresh-water algae. Aside from the fascination of producing freakish plants, experiments with gibberellic acid also provide the amateur with the opportunity of gaining experience with the scientific method-of forcing answers from nature with a minimum of guesswork.

"The experiment which follows is designed to demonstrate certain basic reactions of plants to gibberellic acid. The approach is not limited to this compound; it will prove equally effective with any substance suspected of being a growth stimulator or inhibitor. Thus it can serve as a steppingstone to other investigations.

"You will need a quantity of the acid, a set of identical plants, some inexpensive apparatus and, last but not least, a notebook. Gibberellic acid is sold under the name of Brellin 10 (order No. 450A590 ) by General Biological Supply House, 8200 South Hoyne Avenue, Chicago, Ill. A 28-gram bottle ($2.95) will be more than sufficient for this experiment. A convenient plant for the experiment is the common garden pea, although many other plants are equally rewarding. It should be said, however, that gibberellic acid is known to have no effect on the white pine, the gladiolus or the onion.

"The modern scientific method requires that at least two plants be used in the experiment. One, called the control, is treated with tap water. The other, the experimental specimen, is treated with dilute gibberellic acid. B comparing the subsequent reaction o the experimental specimen with the control the experimenter draws conclusion about the effect of the acid. The method requires that the experimenter make all comparisons on the basis of precise measurements. The elements selected for measurement may consist in the height of the plant, the number of its leaves and its weight. The selection of these particular elements for measurement is not dictated by any hard and fast rule, so the experimenter may choose others But once selected, the elements should not be changed during the experiment. Less obvious effects should also be selected for observation, such as the rate at which the plant consumes oxygen and the percentage of water in the plant with respect to the percentage of organic matter and inorganic ash. Again no hard and fast rules apply. Other factors may be selected for observation.

"We suggest the use of 32 germinated peas as experimental plants. It is well to place 45 or 50 seeds in a germinating medium, which may consist of thoroughly moistened filter paper, a few layers of cotton cloth, or wet sawdust. The medium should be kept warm and should be covered with an inverted glass bowl to prevent evaporation. Some of the seeds may not germinate, but virtually all of those that do will mature. Do not use ungerminated seeds. This experiment seeks to measure the effects of gibberellic acid on a growing plant, not its influence on the sprouting time of seeds. The latter experiment can be equally fascinating and, incidentally, it is one which is of great interest to commercial growers.

"While the seeds are incubating, the experimenter should start his notebook. Every detail, however self-evident, should be entered along with the entry date. It is well to reserve the first few pages for a running summary, including the date on which the acid was received (for information in case the compound should deteriorate with time), the date on which the peas were set for germination, the temperature, the date of planting, when the acid was first administered, the date of the second treatment and so on. It is in this information that explanations will ultimately be found of how the acid does its work.

"When the peas have germinated, 32 paper drinking cups are filled to within a quarter-inch of the rim with sifted topsoil moistened just enough to form a fragile lump when a pinch of it is squeezed. One sprouted pea is then planted in each cup with the tip of the shoot pointing up and flush with the surface of the soil. The cups are arranged in groups of four and labeled according to the concentration of gibberellic acid the group is to receive. One group of four cups is reserved as a control and receives only tap water.

"The gibberellic acid comes mixed with an inert filler. If the experiment were ideally controlled, another group of cups would be reserved for treatment with the filler alone. For this experiment, however, we assume that the manufacturer's filler is really 'inert.'

"A set of seven small bottles with a capacity of three or four fluid ounces will be required for storing dilutions of the acid. These may be purchased for a few cents at most drugstores. The kind which has a scale of cubic centimeters molded in the glass is convenient. If bottles with such scales are not available, the experimenter must either make or buy a graduate.

"The dilutions are prepared by first dissolving one teaspoon (2.5 grams) of Brellin 10 in 50 cubic centimeters of tap water. Each teaspoon contains five milligrams of gibberellic acid; hence each cubic centimeter of this solution will contain a 10th of a milligram of acid. The second dilution is prepared by pipetting 5 c.c. of the first dilution into another container and adding enough tap water to make 50 c.c. Each cubic centimeter of this solution contains one 100th of a milligram of acid, and each dose of 10 c.c. contains a 10th of a milligram. A dilution containing one 100th of a milligram per 10 c.c. is made by pipetting 5 c.c. of the second dilution into a third container and adding water to make 50 c.c. The process is continued until seven dilutions are prepared so that I0 c.c. of each contains, respectively, 1 milligram, .1 mg., .01 mg., .001 mg., .0001 mg., .00001 mg. and .000001 mg. of the acid. Each bottle is labeled to indicate the dilution it holds, and is refilled only with the dilution for which it is marked. Fresh dilutions must be prepared for each treatment, because the acid gradually loses its activity in solution.

"To eliminate the influence of variations in environment during the experiment, the growing plants should be placed in a dark room in which the temperature does not vary appreciably from 70 degrees Fahrenheit, and should be exposed daily for 11 hours to a fluorescent lamp of at least 40 watts placed lengthwise above the cups at a height of two feet. Each experimental plant receives 10 c.c. of the appropriate acid dilution, as indicated by its label, every 48 hours. No water or other solution should be administered to them. The four control plants receive 10 c.c. of tap water at the same time. The acid should always be administered in a uniform manner. Ideally it is applied as a spray by means of an atomizer, which assures that all the exposed parts of the plant receive the solution. If desired, however, the dilutions may be poured on the soil near the base of the plant. Reaction between the acid and the soil tends to lower the activity of the acid somewhat.

"The height of each plant is recorded daily, beginning with zero inches on the day the experiment starts, when the shoots are flush with the top of the soil. A table is ruled in the notebook with a column for recording the height of each plant. From these data graphs may be plotted either for the individual plants or as averages of the groups. Similar tables should be constructed for the remaining indices of growth, such as the number of leaves. Weight need not be measured daily. This is a tedious operation. But the plants should be weighed individually at the conclusion of the experiment.

"An adequate vessel can be made by upending a two-gallon tin can on a plate of flat metal. Hose connections can be introduced through the metal bottom. After the plant and calcium chloride are in place, the assembly is made airtight by applying a ring of wax (made of equal parts of beeswax and rosin) between the can and plate as shown in the accompanying drawing [top]. The wax, which should be applied smoking hot with an eyedropper or a small brush, adheres strongly to cold metal. It may be scraped off at the end of the measurement and reused. One hose connects the vessel with a U-shaped length of glass tubing which serves, when partially filled with colored water, as a manometer for measuring the difference in pressure between the closed vessel and the room. A second hose terminates in a calibrated hypodermic syringe by which air is admitted to the vessel in measured amounts. The third hose serves as a vent for the vessel and is normally kept closed by means of a pinchcock. The potted plant may be supported on a sheet of stiff wire screening placed over the dish of calcium chloride. Unless the seams of the can are airtight they should be coated with wax. A coating of Vaseline will seal the piston of the syringe to the walls of the cylinder.

"To make a measurement, a fresh supply of calcium chloride is placed in the dish, the plant is set on top of the screen and covered by sealing the tin can in place. The piston of the hypodermic syringe is placed at the 10 c.c. graduation. The pinchcock is opened until the columns of colored water in the arms of the manometer stand at the same height, which indicates that the air pressure in the vessel and the room are equalized. The pinchcock is then closed and the time is recorded. After an interval which depends on the size of the plant and the rate of its respiration, the plant will take up enough oxygen from the air to cause an appreciable drop in the pressure indicated by the manometer. The piston of the syringe is then pushed in until the columns of water in the arms of the manometer again stand at the same level. The time is then recorded along with the volume of air admitted to the vessel from the syringe. The volume of air required to equalize the pressure is proportional to the oxygen consumed during the interval, and is an index of the metabolic rate of the plant. The rate of oxygen consumption is computed by dividing the volume of air admitted from the syringe into the vessel by the elapsed time in minutes. Accuracy can be improved by making three successive tests of the same plant and averaging the consumption rate of the three runs. The experiment may extend over several weeks, during which atmospheric conditions as well as the temperature change; for this reason tests made on different days may not be comparable. It is therefore necessary to add a correction which adjusts the figure to the value it would have if the test were made at 'standard' temperature (20 degrees centigrade) and barometric pressure (760 millimeters of mercury). To make this adjustment, the average amount of air admitted from the syringe to the vessel is multiplied by the barometric pressure ( measured in millimeters of mercury) and divided by 760. This quotient is then multiplied by 273 and divided by 273 plus the temperature of the room in degrees centigrade. The result is entered in the notebook for the plant and dated.

"The oxygen consumption of plants varies as a logarithmic function of their surface area. Hence, for strictest accuracy, only plants of identical surface area can be compared. It is not easy to determine the surface area of a plant, but by making the assumption that all plants of equal height have comparable surface areas one can make comparisons which are interesting and useful even though approximate. We have assumed in this experiment that all the plants which are three inches high have the same surface area. The oxygen consumption of the group receiving the highest concentration of gibberellic acid is measured when these plants reach a height of three inches. A day or so later the next group will have reached the same height and can be similarly measured. The test is repeated as each of the more slowly growing groups reaches the height arbitrarily selected. The rate of consumption is plotted (on the vertical coordinate of a graph) against the logarithm of the dose (on the horizontal axis). The logarithm of one milligram per day equals zero, the log of ;1 mg. per day equals -1, of .01 mg. per day equals -2, and so on.

"At the conclusion of the growth period, when, say, the most slowly growing group reaches a height of three inches, all the plants are carefully removed from the paper cups and gently agitated in a large pan of water until the soil adhering to the roots sinks to the bottom of the pan. Care should be taken not to tear away any of the roots. The plants are then rinsed, blotted dry and promptly weighed. Any delay may introduce error because the plants will lose water through evaporation. The weight of each plant is recorded. Then the leaves and roots are cut from the stem and weighed separately. The combined weight of the parts should nearly equal the total weight of the plant. Evaporation may account for a slight difference, but any substantial disagreement may indicate an error in procedure. Graphs of the weight are then drawn, in which the total weight is plotted against the logarithm of the dose, as in the case of oxygen consumption. Similar graphs should be made showing the percentage of weight as a function of dose represented by the leaves, stems and roots respectively.

"Most plants consist mainly of water, and it is important to discover if the growth induced by gibberellic acid represents an increase in the solids or merely an increase in water. This can be determined by thoroughly drying the plants and comparing the dry weight with the total weight previously recorded. Separate tests should be made of the leaves, roots and stems to disclose the effects of the acid on the several parts of the plant. Plants may be dried in about three days by placing them on top of a radiator or under a 100-watt incandescent lamp. To find the percentage of water, multiply the dry weight by 100, divide by the wet weight and then subtract the quotient from 100.

"How does gibberellic acid affect the rate at which plants take up inorganic substances? This can be investigated by burning the dried remains and weighing the ash. In making this test it is again interesting to measure the leaves, stems and roots separately. An accurately weighed sample is placed in a crucible supported in the Hame of a Bunsen burner [see illustration in Figure 4]. Set the crucible somewhat obliquely in an asbestos triangle to allow for the expansion of the heated parts, and close it with a loosely fitted cover. The crucible is then maintained at a red heat until the contents turn to a white, powdery ash. When cool, the ash is transferred to a balance and weighed. Multiply the weight of the ash by 100 and divide by the weight of the dried material placed in the crucible. This gives the percentage of inorganic ash.

"From the accumulated data it is now possible to answer the following questions: Does gibberellic acid increase the rate at which the experimental plant stores energy or merely cause it to absorb an abnormal amount of water? How does gibberellic acid affect the plant's consumption of oxygen? What further experiments do the results suggest?"

"About 10 years ago," he writes, "I participated in a glow-tube experiment with A. R. Perl, A. J. Savard and C. S. Brandt of San Diego. Our tube was constructed with rubber stoppers and sealed with vacuum grease. We were interested in examining discharge tubes as a possible means of producing very high-speed streams of gas to study flows at extreme velocities. To obtain high positive-ion currents we inserted a little booster discharge at the anode end of the tube, hoping this would create more local ions. The apparatus is depicted in the accompanying drawing.

"Of more interest to other experimenters than the ion booster, however, may be the simple and inexpensive vacuum pump we used. Falling drops of mercury were employed as multiple pistons to entrap the gas and force it from a capillary tube. By adjusting the pinch clamp on the hose connecting the upper reservoir to the capillary tube the mercury can be made to fall as discrete drops. Our capillary had a bore of about two millimeters. The manometer used to measure the pressure of the system was a simple U-tube which we inclined at a slope of 10 to 1 to increase the range through which the mercury moved with changes in pressure. It is necessary to tap the manometer gently to obtain good equilibrium levels and to 'break' the mercury away from the sealed end of the U-tube. Incidentally, serious error will result if any gas other than mercury vapor remains in the manometer. Some effort was made to eliminate such gas by heating the sealed end of the U-tube gently when the system was at low pressure. I hasten to point out, however, that this is a tricky and somewhat dangerous operation, because if the tube should break, highly poisonous mercury vapor would be released.

"For a power supply we used two 110and 440-volt transformers with the primary windings in parallel and the secondaries in series. The secondary output of 800 volts was converted to direct current by means of a full-wave rectifier which used four 866A vacuum tubes, as shown in the drawing. The variable-shunt resistor connected across the ends of the glow tube supplies voltage to the auxiliary discharge plates and has a resistance of about 100,000 ohms. At a rating of only one watt it was somewhat overloaded because it maintained some 400 volts across itself. The ballast resistor in series with the glow tube was a makeshift affair and a source of some difficulty. We tried wet resistors, using various concentrations of contaminant in water to vary the resistance, and were about to try a bank of resistors made o Christmas-tree lamps when our attention was diverted to other matters. Perhaps this is the 'weak link' in the setup. We did produce some mighty pretty glows however, with all the characteristics shown in textbooks."

Bibliography

PLANT GROWTH SUBSTANCES. Frank B. Salisbury in Scientific American, Vol. 196, No. 4, pages 125-134; April, 1957.

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