A TECHNIQUE FOR THE CULTIVATION OF HIGHER PLANTS UNDER STERILE CONDITIONS

HENRY R. KATHREIN

(WITH

TWO

FIGURES)

Received February 12, 1951

To study the effects of microorganisms of the soil on plant growth, it is essential that methods be developed by which plants can be grown under sterile conditions. FRED (2) has reported the use of a modification of KELLERMAN 's (5) system by which peas were grown in soil under sterile conditions. This method entailed the use of a glass cylinder which fitted onto a specially constructed metal pot in which the plant was to be grown. A glass beaker was inverted over the cylinder to prevent contamination. In a method developed by GERRETSEN (3, 4) oats, mustard, and sunflowers were grown in quartz sand under sterile conditions. Sterility of the pots was maintained by covering the sand with a layer of sterile cork filings mixed with katadyne silver. In both methods described, the nutrient salts were added directly to the pot before sterilization. REUSZER (6) has described a method of cultivating squash and sunflowers under sterile conditions. In this method, the seed was placed in a vermiculite plant tube and the root allowed to grow down into a chamber containing a nutrient solution. A similar technique for growing plants with their roots in a sterile medium has been presented by BLANCHARD and DILLER (1). This method involved the placing of the seed in an aluminum column filled with vermiculite. Upon germination, the root penetrated a gauze layer into a sterile nutrient solution. Studies on the nutrition of plants are being conducted in this laboratory by the use of sand culture. The plants are watered with nutrient solutions of known composition and flushed periodically with distilled water to prevent salt accumulation. None of the above methods were completely applicable to this method of raising plants; therefore, the technique described in this paper was developed. This technique involves the culturing of higher plants in pure quartz sand in such a manner that the entire plant is held under completely sterile conditions until maturity is reached. Seeds were sterilized in a small filter flask using a 0.1%o mercuric chloride solution. The period of sterilization varied with the type of seed to be sterilized; in the case of oats, immersion for a four minute period was sufficient to destroy the microorganisms present. During the sterilization period, a vacuum was alternately applied and released; and the flask was shaken to remove air bubbles from the surface of the seed. The seeds were then washed four or five times with sterile distilled water to remove any trace of the mercuric chloride. Following this treatment, the seeds were transferred to a bacteriological agar and allowed to germinate. Contamination was checked 843

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by the sterility of the agar medium on which the seeds were placed. All seeds which showed contamination were discarded. When the roots of the plants reached an elongation of approximately five mm., the seeds were transferred aseptically with forceps to the sterile pots of sand. Details of the apparatus described below are shown in figure 1. A standard four inch glazed soil jar with a hole in the side near the base was used

SCREW

COTTON PLUG
-100 MM. GLASS CYLINDER ALUMINUM WATERING TUBE

PARAFFIN

SOIL
JAR

)LLECTION
FIG. 1. Apparatus used for the culturing of higher plants under sterile conditions.
as a culture chamber. A rubber stopper with a glass tube passing through it was inserted into the hole at the base of the pot. To permit the drainage of excess nutrient solution, this glass tube was connected to a 1000 ml. Erlenmeyer flask by means of rubber tubing. A glass cylinder 100 mm. in diameter and 400 to 600 mm. in length and stoppered at the top with a large cotton plug was fitted onto the top of the soil jar and held in place by a paraffin seal. A piece of 1/4 inch aluminum tubing wound tightly between the layers

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of the cotton plug was passed down the cylinder into the soil jar. The aluminum tubing was made with a three inch loop on the soil jar end with holes drilled at one inch intervals on the loop. This tubing allowed the plant to be watered directly at the surface of the sand without washing the sand from around the roots of the plants. The top of the aluminum tubing was connected to a bottle of sterile nutrient solution and a bottle of sterile distilled water with rubber tubing. This allowed the soil jars to be watered with nutrient solution and flushed with distilled water. The bottles used for nutrient solution and distilled water were 20-liter aspirator bottles. Any number of soil jars may be connected to one nutrient solution bottle and one distilled water bottle by means of glass T connections. Circulation of air inside the glass cylinders may be accomplished by adding another T connection and forcing air through a cotton stoppered tube into the aluminum

watering tube. The soil jars containing the quartz sand were covered with a layer of gauze, cotton, and kraft paper tied in place. The rubber connections of the drainage tubes at the bottom of the jars were also wrapped with gauze, cotton and paper covers. The jars were then sterilized in an autoclave for three hours at 1210 C, 15 pounds steam pressure to insure complete sterilization. The bottoms of the glass cylinders were covered with gauze, cotton, and paper wrappers tied in place. All other connections of the apparatus were covered with gauze, cotton, and paper wrappers to maintain sterility after autoclaving. The glass cylinders, drainage bottles, and all T connections were autoclaved 15 minutes at 15 pounds of pressure. Because of their size, the 20-liter aspirator bottles which were used for nutrient solutions and distilled water were sterilized by dry heat (1800 C, one and a half hours). The top and bottom openings were plugged with cotton. Following the sterilizing process, these plugs were replaced with rubber stoppers which had glass tubing connections. The stoppers and connections were sterilized in an autoclave. The nutrient solutions and distilled water were sterilized by passing them through a size A, normal porosity Berkefeld filter. After the seeds had been sterilized and the proper stage of germination reached, they were transferred aseptically to the soil jars. The paper, cotton, and gauze wrapper was loosened from the top of the soil jar; and the seeds were positioned in the sand by means of sterile forceps. The wrappings were then removed from the glass cylinder and jar, and the cylinder was placed on top of the jar. The cylinder was sealed to the jar immediately with sterile paraffin. The paraffin was applied with a half inch brush that had been sterilized with the paraffin in a small Erlenmeyer flask. The aluminum tubing emerging from the cotton plug at the top of the cylinder was then connected to the nutrient solution and distilled water bottles by removing the wrappings and making the connection. The connection from the bottom of the soil jar to the collecting Erlenmeyer flasks was made in the same manner. The apparatus was then completely assembled, and the seeds were watered with distilled water. The nutrient solution and distilled water

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FIG. 2. Oat plants which have been grown under completely sterile conditions.

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were controlled by means of screw clamps which enabled the watering with nutrient solution or the flushing of the jars with distilled water. Sterility of the soil jars was checked periodically by removing the drainage bottles and plating the nutrient solution with various bacteriological media.

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Complete sterility was maintained on oat plants over a period of eight weeks after which time they were harvested for analysis. Average height of these plants was 500 mm. Figure 2 shows a set of oat plants raised under sterile conditions by the above method.
THE SAMUEL ROBERTS NOBLE FOUNDATION ApDMORE, OKLAHOMA

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LITERATURE CITED BLANCHARD, FRED A. and DILLER, VIOLET M. Technique for growing plants with roots in a sterile medium. Plant Physiol. 25: 767-769. 1950. FRED, E. B. The growth of higher plants in soil free of microorganisms. Jour. Gen. Physiol. 1: 623-629. 1919. GERRETSEN, F. C. Das Katadyn Verfahren zur sterilen Kultur hoherer Pflanzen. Planta 23: 593-605. 1935. GERRETSEN, F. C. The influence of microorganisms on the phosphate intake of the plant. Plant and Soil 1: 51-81. 1948. KELLERMAN, K. F. U. S. Dept. Agr., Bur. Plant Ind., Bull. 120. 1914. REUSZER, HERBERT W. A method for determining the carbon dioxide production of sterile and non-sterile root systems. Soil Sci. Soc. Amer. Proc. 14: 175-179. 1949.