GROWTH STIMULATION BY MANGANESE SULPHATE, INDOLE‐3‐ACETIC ACID AND COLCHICINE IN POLLEN GERMINATION AND POLLEN TUBE GROWTH

DUE TO simplicity in structure, readiness to grow in controlled conditions, and sensitivity to environnental factors, pollen has been a favorite for physiological studies. Though pollen grains can germinate and grow readily in water or in sugar media without external supply of nutrients, their rate of growth can be accelerated by the crushed tissues of stigma or their extracts (Knowlton, 1922; Sasaki, 1925; Noguchi, 1931), by sterilized yeast (Brink, 1924a), by vitamins (Dandliker, Cooper and Traub, 1938; Cooper, 1939), and by growthpromoting substances such as indole-3-acetic acid (Smith, 1939). Recently the beneficial effect of micro-elements such as boron on the growth of pollen grains has been demonstrated (Cooper, 1939; Bobko and Zerling, 1938; Blaha and Schmidt, 1939). It is considered of interest to study whether the presence of manganese sulphate has any effect on pollen germination and pollen tube growth and whether or not this effect is similar to that of indole3-acetic acid and colchicine. Experiments with several kinds of test plants were conducted during 1941-1942, the results of which will be described in the following paragraphs. MATERIALS AND METHODS. -Pollen grains of several kinds of plants were used, namely: Antirrhinum majus L. , Nicotiana tabacum L. , Thea sinensis L. , Allium cepa L. , A. flstulosum L. , A. scorodoprasum L. var. viviparum Regel, A. odorum L. , Brassica juncea Coss. , Triticum vulgare Vill. , Hordeum sativum Jess. , Zea Mays L. , and Oryza sativa L. MIaterials were obtained from the Agricultural Experiment Station of the University. The pollen grains were collected at eight to nine o'clock in the morning of a fine day in the blooming period. They were stored in a Petri dish which was placed in a desiccator. At the beginning of an experiment, one drop of the freshly prepared medium was placed on a cover glass made of non-alkaline glass and a small number of pollen grains were dispersed in the medium with the help of a platinum wire. The cover glass was then mounted on a concave slide, the margins of the cover glass being smeared with either vaseline or paraffin. In the case of cereals, collection of pollen grains was secured in the following manner. The upper part of the lemmas was cut with a pair of small scissors. One or two hours later, stamens elongated out of the lemma. The pollen grains could be collected in a watch glass by simply agitating the spikelets with a glass rod. With a clean Chinese brush of wool, the pollen grains 'Received for publication December 7, 1943. Manuscript received through the Department of State, Washington, D. C. , and published under a special ruling of the Editorial Board. could be easily transferred and dispersed in the hanging drop. After an interval of time, which varied according to the species used, the preparations were observed under the microscope. More than 100 pollen grains were observed and the percentage germination was calculated on the basis of these observations. The measurement of ten pollen tubes chosen at random was taken with ocular micrometer either at the same time or after an interval. The operations were repeated four times in each experiment and the average values were recorded. Thus over 400 pollen grains were counted and more than forty pollen tubes were measured in each test. Four kinds of culture medium were used in these experiments, namely: (1) manganese sulphate, (2) indole-3-acetic acid, (3) colchicine, and (4) the control. With few exceptions, the basic medium which served as the control was a sucrose or a glucose solution whose optimal concentration for pollen germination was determined previously by experiment. To this basic medium, manganese sulphate, indole3-acetic acid and colchicine were added, respectively, in a series of dilutions from 10-4M. to 10-10M. In some cases, sucrose-gelatin, glucose-gelatin, sucrose-agar and redistilled water were also used as the basis of culture media. Manganese sulphate used in these experiments was a guaranteed reagent of E. Metek, but colchicine and indole-3-acetic acid were obtained from Mallinckrodt Chemical Works, U.S.A. All the utensils and the culture media were strictly sterilized before use. Temperature was controlled during experiment: in cases of long duration, the preparations were placed in an incubator for germination and growth. Brink's hanging drop method was followed in experiments except in cases where sugar-agar was used as the basis of culture medium. RESULTS. -1. Antirrhinum majus L. -Knowlton (1922) found that the Antirrhinum pollen germinated readily in a basic medium of sucrose-agar with a small amount of crushed stigma. In the present work, 0.35 M. sucrose as well as 0.35 M. sucrose in 1 per cent agar were used as the basis of the medium. Pollen germination and pollen tube growth were determined after four hours culture. Tables 1 and 2 show the results of these experiments. Owing to the variation of temperature during this interval, percentage germination of the control showed a small fluctuation. Indole-3-acetic acid caused an increase in germination of about twenty per cent over the control in an optimal concentration of 10-6M. and this stimulating effect increased with concentrations within the range of 10-10 10-6M. A concentration of 1o-5M. , however, was less effective than 10-6M. The effects of manganese sulphate and col-