ON THE QUESTION OF OCCURRENCE OF INDOLE‐3‐ACETIC ACID IN PINUS SILVESTRIS

The inner bark of Pinus silvestris L. contains a natural acidic growth promoter which stimulates elongation of wheat coleoptile sections and oat mesocotyl sections, produces oat coleoptile curvature, and is not effective in an oat first-leaf increment test. It occupies the same or a closely related RF to that of authentic IAA if rechromatographed with seven partition solvents. Pine auxin described by Fransson could not be detected by spraying with reagents known for developing the color reaction of IAA. Natural pine resin from wounds, when added to test solutions, affects several characteristic features of authentic IAA which are commonly used for its identification. These are: (a) position on chromatograms (RF), especially if 70% ethanol is used as the partition solvent, (b) amount of growth promotion in bioassays, and (c) ability to develop specific color reactions with IAA on chromatograms with known reagents. Interaction with pine resin or other related substances which could not be separated from the extract by conventional partition methods may be responsible in previous studies for the difficulties in identifying the auxin of Pinus silvestris as an indole-3-acetic acid. NATURAL growth-promoting substances have been known for many years to occur in different species of pines. These substances have been found in buds, shoots, and inner bark mostly by diffusion into agar blocks and bioassayed by Avena coleoptile curvature tests (Czaja, 1934; Zimmermann, 1936/7; Mirov, 1941; Onaka, 1942, 1950; Brown and Wetmore, 1959), or bv paper chromatography of extracts and coleoptile straight-growth test (Ne6esany, 1958; Krugman, 1965). An important approach to the identification of these substances as IAA was provided by studies using specific color reactions for detection of indole compounds (Westing, 1960) and comparison of the RF region they occupied with that of authentic IAA (Allen, 1960; Larson 1962, 1963; Ogasawara, 1961a, b). Fransson (1953, 1959), however, performed a series of experiments with auxin extracted from the shoots of Pinus silvestris L. and found it not to be indole3-acetic acid. In addition to differences in their diffusion coefficients in agar, he also found that pine auxin occurred at a different RF from that of IAA chromatographed with 70% ethanol. Received for publication 12 October 1967. This work was supported in part by a grant from the U. S. Department of Agriculture (No. FG-Po-166) to the Department of Forest Botany, Central College of Agriculture, Warsaw, Poland. The paper was prepared while the author was a Charles Bullard Fellow at Harvard University, Cambridge, Mass. The author wishes to express his gratitude to Professor Helena Birecka and to Professor Kenneth V. Thimann for giving him a chance to discuss with them the questions under investigation. He thanks Dr. L. Witkowska-Zuk for helpful assistance in the various bioassay techniques. He is also indebted to Miss A. Grodek and Mrs. G. Karczmar for very valuable and generous technical assistance in these experiments. This paper presents the results of chromatographic investigations of a natural growth promoter found in methanolic extracts from the inner bark of Pinus silvestris collected from the trunks of 60-year-old trees at different intervals during the growing season. It represents a part of some wider studies on the role of natural growth substances in the differentiation of an annual ring of wood in this species. M\ATERIAL AND METHODS-Tissues outside the wood cylinder, comprising cambium, phloem, and periderm (except dead cork) were collected from the trunks of trees growing in Experimental Forests in Rogow during June, July, and September. The samples of tissue were immediatelv frozen with solid C02, weighed, and stored at -25 C until extracted. Pine resin was collected as exudate from wounds made in the trunks of the same trees at the same time. Extraction--Extraction procedures were similar to those described by Phillips and Wareing (1958). Known amounts of tissue were homogenized with ice-cold absolute methanol (MeOH) in a Waring Blendor and extracted overnight at 0 C. After 16 hr, water was added to obtain a final concentration of 80 % MeOH, and the tissue was extracted for an additional 2 hr. The extract was filtered, and the residual tissue extracted with two additional aliquots of 80% MeOH. The three extracts were combined and evaporated to dryness under vacuo at 8 C. The residue was taken up into a known volume of 80% i\JeOH and purified by solvent partition. Purification--The 80 % aqueous MeOH extract