Free Auxins and Free Tryptophane in the Strawberry.

Abstract

It has been previously demonstrated (15) that the growth of the strawberry receptacle is regulated by the achenes. This control is exerted through substances which are active in the Avena test, a standard test for auxins. The exact identity of these natural auxins, however, had not been investigated. The purpose of this paper is to report further experiments on the production of auxins by strawberry achenes, on the level of free tryptophane, a generally presumed raw material in the genesis of natural indolic auxins, and on the separation of the auxins of the strawberry by paper chromatography. MATERIAL AND METHODSStrawberries, var. Marshall,3 were grown in pots in the greenhouse and hand pollinated. The fruits were removed at three day intervals during a period extending from 3 to 30 days after pollination, at which time the berries were ripe. Upon picking, the fruits were immediately brought to a cold room (50 C), the achenes were separated from the receptacles, both fractions were frozen by placing in a deepfreeze, and the frozen material was lyophilized. The lyophilized samples were used for auxin and tryptophane determinations.Auxins were extracted with peroxide-free ether (water saturated) alone, or with a mixture of one part of acetone and two parts of ether (V/V) in an ice-bath at 0°C, in the dark, for 2 to 4 hours. It is generally agreed that such a procedure yields only "free" auxin, by contrast to treatments with alkali, proteolytic enzymes, or merely prolonged incubation at room temperature (29)."Free" tryptophane was extracted according to the procedure of Dr. J. H. M. Henderson (unpublished) which consists of two steps: (1) the precipitation of proteins by boiling ethanol for 3 minutes, followed by the evaporation of the alcohol on a steam bath; (2) the extraction of tryptophane with hot water (repeated twice). The aqueous extract thus obtained was shaken with ether at pH 4.0 to purify it from possible traces of indole and anthranilic acid which give a positive test in the bioassay used. Then, the pH of the aqueous extract was adjusted to 6.0, and its tryptophane content measured quantitatively by the Lactobacillus assay (16, 17), a procedure which (in the absence of indole and anthranilic acid) is remarkably specific for L-tryptophane, as recently confirmed by Stowe (22). Brighton gave a satisfactory Avena test. For the straight growth bioassay, seeds were soaked for two hours in water and, then, laid down on wet facial tissue through which roots can grow freely (which is not the case with filter paper). The seeds were placed for about 4 hours at approximately 35 cm under a 32 watt G.E. white fluorescent "Circline" light filtered through a Corning filter No. 2404, to suppress growth of the mesocotyl. After the red light treatment, the seedlings were grown in a darkroom at 25°C and 85 % relative humidity. When the coleoptiles reached about 2.5 cm in length, the first 3 mm from the tip were cut off and discarded, while the next 4 mm were saved for the assay. Four mm rather than 10 mm sections were us...

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