Gibberellin Response in Lettuce Hypocotyl Sections

Abstract

Excised lettuce (Lactuca sativa L.) hypocotyl sections retain the ability to elongate in response to gibberellic acid (GA3) addition. In 48 hr at 30 C a GA3-treated segment more than doubles while a control segment elongates less than 50%70. Auxin has no detectable effect on this system. Sensitivity to GA3 is not decreased by apex or root removal. Of the experimental variables tested, temperature, sucrose, and preincubation in water affect growth both with and without GA3. Blue and far red light inhibit growth without GAs; this inhibition is reversed by GA3. Potassium chloride stimulates growth of illuminated sections treated with GA3 but has no effect on control growth. When sections are incubated in the dark, KCl has a promotive effect on elonga-tion. There are numerous advantages to using excised plant segments to study the mechanism of hormone action. Interactions, such as transport of hormones and nutrients to the responding segment, are eliminated as variables. The recent advances which have been made in the characterization of the auxin response clearly demonstrate the utility of such isolated segments (4, 5). Research on the mechanism of gibberellin-induced elongation has been hampered by an apparent diminution in ability to respond to GA3 when stem segments are removed from meriste-matic regions (10). Recently, Kaufman and his colleagues (9, 13, 14, 19) demonstrated a dramatic effect of GA3 on growth of excised stem segments of Avena, but sections from dicotyledonous plants have seemed less responsive. Brian and Hemming (1) showed that the growth of segments from dwarf pea stems was much less than that realized by similar regions of whole plants and was not elevated by GA3 application. Furthermore, the GA3 response of many isolated sections has been shown to be dependent on added auxin (1, 27, 30) or sucrose (24). This paper describes the GA3-induced growth of sections isolated from the hypocotyl of lettuce seedlings. MATERILS AND METHODS Plant Material. Seeds of lettuce (Lactuca sativa L. cv. Arctic) were obtained from Carter's Ltd., Raynes Park, London. Seeds were imbibed for 2 hr in an excess of distilled H20 and were sown in white light in 9-cm Petri dishes on a circle of Whatman No. 1 filter paper wetted with 5 ml of distilled H20. Germination proceeded in the dark at 25 C for 34 hr after which time the 1 This work was supported by Grant GB 27468 from the National Science Foundation. seedling hypocotyl was 2 to 2.5 mm. Sections were excised from the hypocotyl using a dissecting microscope. The lower cut was made above the swelling which marks the transition zone, and the apical region and cotyledons were removed at a point below the juncture of the cotyledons. For convenience, the excised sections were floated in water at 3 C for 1 hr before incubation with GA3. Incubation. Unless otherwise noted, hypocotyl sections were incubated at 30 C in 7-cm plastic Petri dishes containing a circle of grid filter paper (Schleicher and Schuell, No. 9) and 2 ml of incubation medium (Fig. 1). Illumination was from a bank of four Sylvania "Daylight" fluorescent tubes suspended above the dishes and providing an intensity of 300 ft-c incident on each dish. Sections were incubated in distilled H20 and the appropriate concentration of GA3 (K salt GA3, Sigma Chemical Co.) or other additive. Growth Measurement. The growth of sections was determined by careful measurement of photographic images (Fig. 1). The incubation dishes were placed on a Plexiglas shelf which was situated above and parallel to a set of tracks along which a modified copy stand could be wheeled. The copy stand was fitted with a close-up lens (Beseler Macro Auto-Topcor, 1: 3.5, f = 58 mm). Columnated light was provided by a microscope lamp mounted on a hinged arm in a swivel socket. To cast an image of the growing hypocotyl sections, overhead lights were extinguished, and the microscope lamp was positioned over the Petri dish. The copy stand was wheeled under the light cone, and the lens was used to focus the images onto the copy stand. Images were recorded by exposing a sheet of photographic paper. Images could also be recorded using a camera mounted on the copy stand at the position of the close-up lens. Using this apparatus it was possible to monitor growth in 15 dishes concurrently without mechanically disturbing the growing sections. Growth in the vertical plane was not detectable by this method, and it was occasionally necessary to manipulate sections onto their sides to obtain accurate images. Analysis of data. Photographic images produced by the method described above were measured with a map reader (Keuffel and Esser, Switzerland). Both sides of each image were measured, and the average of the measurements was termed length. Differences in enlargement were compensated by measuring the square grids of the filter paper image in each case (Fig. 1). In the first photograph of a time sequence, the hypocotyls were identified by position, and length changes in subsequent photographs were computed separately for each hypocotyl. In this way percentage length change could be computed as the average of 10 individual percentage length changes. The expression %AL denotes average percentage length change and is defined by the equation 100 N (Ln)fina1-(Ln)initial N n_1 (Ln)initial where N is the number of hypocotyls and L is length. This index is not constant in time or uniform along the hypocotyl, but in a constant time interval it provides a rough measure of growth. 267