Cotyledon Expansion as a Bioassay for Cytokinins

Abstract

The activity of cytokinins in stimulating cell division formed the basis for the original techniques for their bioassay (12, 13) and for later modifications (6,7,8). In the subsequent decade, as various other activities of cytokinins blecame evident, numerous other types of stimulations have been employed in bioassays. These include cytokinin stimulations of Lemna frond growth (2), of seed germination (11), of leaf enlargement (4), or the cytokinin deferral of leaf senescence (9). Each of .these assay techniques has limitations, being relatively complicated, utilizing relatively large volumes of solution, requiring relatively lbng time periods as in the case of the tissue culture assays, or being relatively insensitive or non-specific for cytokinins. We have found that the growth of the cotyledon tissue of Xanthium expresses a large and rapid cytokinin response which can be obtained in very small solution volumes, and which is sensitive to cytokinin concentrations as low as 10-8 M. The procedure for bioassay is as follows. The seeds of Xanthium pensylvanicum Wallr. are removed from the burred fruit using a razor cut along the side of the bur, avoiding damage to the seeds. Selecting either the large (upper) or the small (lower) seeds for any given experiment, three 3 mm sections are cut from the center of the seed, discarding the end pieces. The 3 sections are each cut again longitudinally, and the 6 pieces are placed on wet filter paper for 5 to 7 hr to complete the first phase of water uptake (1). The pieces are then rinsed in distilled water, and separated into the 2 cotyledonary pieces, resulting in 12 pieces of tissue from each seed. Ten randomized pieces are used for each cytokinin determination, and after initial weight of the group of 10 has been taken, each group is placed on filter paper moistened with the solution to be tested. For solutions in generous supply we