Changes in germination inhibitors, cytokinins, and gibberellin-like substances during the breaking of coat-imposed dormancy of sycamore seeds at low temperature suggest that dormancy may be controlled by the presence of endogenous germination inhibitors. Stratification at 5 °C led to a greater loss of neutral inhibitor(s) from the embryo than did incubation at 20 °C. No marked differences were observed in the level of cytokinins and gibberellin-like substances between treatments. However, a gradual decrease in the level of cytokinins was observed during stratification. Whether a cytokinin-inhibitor interaction is involved in the control of dormancy of sycamore seeds, as was previously suggested, remains to be determined. INTRODUCTION Experiments with dormant sycamore (Acer pseudoplatanus L.) fruits indicated that fruits and seeds with the testa intact require a period of chilling at 5 °C to break dormancy, whereas isolated embryos germinated immediately at 20 °C on moist filter-paper without pretreatment (Webb and Wareing, 1972a). Results from leaching experiments suggested that dormancy was the result of the restric tion, by the testa, of the outward diffusion of germination inhibitors present in the embryo. Inhibitors extracted from dormant sycamore seeds have also been shown to inhibit the germination of non-dormant sycamore seeds at relatively low concentrations (Webb and Wareing, 19726). A comparison with the effects of applied abscisic acid (ABA) indicated that the level of endogenous ABA alone could not account for the inhibition observed by the seed extracts. Both ABA and neutral compounds present in the embryo appeared to be involved. Leaching treatments that removed dormancy also led to a decrease in the level of inhibitors present in the basic and neutral ethyl acetate fraction. Application of kinetin to dormant sycamore seeds increased germination 1 Present address : Great Lakes Forest Research Centre, Canadian Forestry Service, P.O. Box 490, Sault Ste. Marie, Ontario, Canada. 2 Present address : Botany Department, University of Natal, Pietermaritzburg, Republic of South Africa. This content downloaded from 40.77.167.104 on Sun, 24 Jul 2016 04:18:58 UTC All use subject to http://about.jstor.org/terms 742 Webb, van Staden, and Wareing—Seed Dormancy in Acer whereas gibberellic acid (GA3) had no effect. A similar response was obtained with lettuce seeds in which germination was inhibited by the basic and neutral ethyl acetate fraction obtained from dormant sycamore seeds. These results led to the suggestion that a possible inhibitor-cytokinin interaction may be involved in the dormancy of sycamore seeds (Webb and Wareing, 19726). Similarly, the work of Pinfield and Stobart (1972) supports this hypothesis. The results of Van Staden, Webb, and Wareing (1972) with Acer saccharum suggested that endogenous cyto kinins may play an important role in embryo-dormant seeds. Stratification led to a marked increase in the level of cytokinins and gibberellin-like compounds with a concomitant loss of ABA. In sycamore seeds which show a coat-imposed dormancy, the natural means of dormancy removal is by moist low-temperature after-ripening as the fruit over winters on the ground. Thus, it was of interest to examine the possible involvement of endogenous germination inhibitors and promoters during the breaking of coat imposed dormancy of sycamore seeds at low temperatures. MATERIALS AND METHODS Seed storage and germination conditions A. pseudoplatanus L. fruits were collected from single, open-grown trees in the Botany Gardens, Aberystwyth, in November 1970 and 1971. The fruits were air-dried to remove surface water only and stored in sealed containers at 2-5 °C. In these studies seeds were used within 2 months from the dated collection since it is difficult to store sycamore fruits in a dormant state without loss of viability (Jahnel, 1955). Seeds were germinated on filter-paper in Petri-dishes at 20 °C (Webb and Wareing, 1972a). Protrusion of the radicle through the covering structures was used as the criterion for germination. Stratification conditions Whole fruits were presoaked in distilled water for 24 h and placed in plastic bags. Any excess moisture was drained off and the sealed bags stored at 5 °C for approximately 80-100 d. At 10-d intervals the bags were opened and checked for moisture. Extraction and bioassay of growth regulators Germination inhibitors and gibberellin-like substances were extracted as previously described (Webb, Van Staden, and Wareing, 1972). The seed material was homogenized and extracted in 80 per cent redistilled methanol at 2-5 °C. The aqueous fraction was partitioned four times at pH 8-0 with redistilled ethyl acetate. The aqueous fraction was then adjusted to pH 2-5 and again partitioned with ethyl acetate. Cytokinins were extracted using the methods of Van Staden et al. (1972). The partitioned extracts were dried under vacuum at 35 °C and strip-loaded on to Whatman No. 1 chromatography paper. The chromatograms were developed in a descending manner in iso-propanol: ammonia iwater (10:1:1 v/v/v) in the case of extracts to be assayed for inhibitors or gibberellins, and in water-saturated sec butanol for extracts to be assayed for cytokinins. Germination inhibitors in the acidic ethyl acetate fraction and in the basic ethyl acetate fraction (which would also contain any neutral compounds) were determined using the lettuce-seed germination bioassay (Webb and Wareing, 19726). In all experiments recorded the results are the means of at least two bioassays seen at different times. The lettuce hypo cotyl bioassay was used to determine gibberellin-like activity (Loveys, 1970). Cytokinin activity was determined using the soybean callus bioassay (Miller, 1965). RESULTS Whole fruits from the 1970 seed crop were stratified at 5 °C and a second control sample was incubated at 20 °C (a temperature that normally does not lead to This content downloaded from 40.77.167.104 on Sun, 24 Jul 2016 04:18:58 UTC All use subject to http://about.jstor.org/terms Webb, van Staden, and Wareing—Seed Dormancy in Acer 743 Fig. 1. The effect of stratification on the level of acidic germination inhibitors in sycamore seeds. The equivalent of 0-5 g dry weight of i material in the acidic ethyl acetate fraction was chromatographed on paper in '10:1:1' and assayed with the lettuce seed germination bioassay. r