THE PHOTODYNAMIC ACTION OF NEUTRAL RED ON ROOT TIPS OF BARLEY SEEDLINGS. PART I. THE EFFECT ON FREQUENCY OF CELL DIVISION

A CYTOLOGICAL study of the photodynamic action of neutral red on the root tips of barley seedlings was undertaken in an attempt to extend the data on this reaction in plants. Most of the investigations on the photodynamic action of dyes have been concerned with animal tissues (e.g., Raab, 1900; v. Tappeiner, 1909; Jodlbauer, 1906; Politzer, 1924; Blum, 1932, 1935; Mencke, 1935; Tennent, 1938; Frankston, 1940; and others). The early work on plants was confined to bacteria, yeasts and molds and has been reviewed by v. Tappeiner (1909). The action of visible light on these organisms immersed in dilute dye solutions resulted in death of the cells, whereas dye alone had no effect. The injury appeared much later in plants than in Paramecium. Gicklhorn (1914) in a series of well controlled experiments found that when Elodea leaves, or entire shoots of Ceratophyllum, were put in fluorescent dye solutions and exposed to light, a photodynamic injury resulted. The initial appearance of the injury, as well as the subsequent death of the cells, always occurred earlier in the light than in the dark. Observations of the course of the injury were made on living leaves of Elodea, shoots of Ceratophyllum, isolated cells of the leaves of phanerogamic land plants, and on the more favorable Spirogyra filaments. No study of fixed tissue was reported. Becker (1929, 1930, 1933, 1935) in the course of his extensive work on the effect of vital dyes on cell division in plants found that onion root tips were photodynamically injured when exposed to light in a dilute solution of methylene blue (1:100,000). His findings from this study of fixed tissue were confirmed by vital observation on the epidermis of young leaves of Allium and the stamen hair cells of Tradescantia. He reported that neutral red in the dark, even in a 1:500 dilution, gave few abnormalities and they appeared later than in the case of methylene blue. No mention of the effect of neutral red in sun or in diffuse light could be found in Becker's papers. Metzner (1923) found that roots of wheat seedlings growing in a dilute solution of erythrosin bent toward the light. Blum and Scott (1933) obtained similar results with wheat seedlings grown in a 1:500,000 erythrosin solution and irradiated in artificial light. They found that the wave lengths of light 1 Received for publication April,14, 1941. Parts I and II comprise a dissertation presented to the Faculty of Bryn Mawr College in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The subject of this investigation was suggested by the late Dr. David H. Tennent and the author is glad to take this opportunity to express her appreciation for his valuable advice and criticism during the course of the work. She wishes also to thank Dr. Mary S. Gardiner and the Department of Biology of Bryn Mawr College for the laboratory facilities placed at her disposal. that gave the phototropic bending corresponded to maxima in the absorption spectrum of the dye, i.e., the dye acted as a photosensitizer. Roots in water alone showed no bending toward the light. Blum and Scott state that "in paraffin sections this differential growth appears as a less degree of elongation of the cells on the illuminated side than on the dark side." They do not report a further cytological study. In view of the above work it seemed desirable to investigate the photodynamic effect of vital dyes on cell division in root tips. Neutral red was chosen, since it is one of the least toxic of the vital dyes. Zea mays seedlings were used at first but were found to be exceedingly resistant to the photodynamic action of neutral red. Root tips were immersed in solutions of neutral red of graded concentration (1:4,0001:150,000) and irradiated in sunlight for one hour. No appreciable effect on the rate of growth was observed. Growth was inhibited in 1:4,000 solutions but this concentration was also somewhat injurious in the dark. Differential counts of cell divisions in roots irradiated in 1:75,000, 1:10,000, and 1:5,000 neutral red showed no deviations from the normal rhythm of the division cycle. Abnormalities of the chromosomes were seen only infrequently in the material irradiated in a 1:10,000 solution and although more were seen in the sections of roots irradiated in 1:5,000 neutral red, the degree of injury compared to that in the controls did not seem great enough to warrant a more detailed analysis. It is interesting that Bersa (1927) found that root tips of Zea mays were also very resistant to X-irradiation. Root tips of Hordeum vulgare seedlings proved to be a material that was much more sensitive to the photodynamic action of neutral red. A detailed account of the experiments on barley will be given in Part I and Part II of this paper. The first part will deal with the results of counts of the dividing cells at various times after irradiation, and the second part will discuss the morphological abnormalities found in the root tips. In general these experiments showed that irradiation for one hour in sunlight in a 1:75,000 solution of neutral red produced complete cessation of cell division in about seven hours after irradiation. Various abnormalities of division were present. The number of cell divisions was normal in controls kept in neutral red in the dark, and only partially reduced in controls irradiated in distilled water. According to the amount of sunlight reaching the roots and the susceptibility of the particular root tips, either division started again in twenty-two or more hours or death ensued. MATERIAL AND METHODS.-Grains of a genetically pure strain of Hordeum vulgare, "six row, high pro-