The uptake of 8-14C1N'-benzyladenine (BA) was studied in excised watermelon (Citrulls vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.There seems to be a correlation between the level of radioactivity (ie. of BA + derivatives) present in the cotyledons and the magnitude of hormonal responses that are observed four days after uptake. This relationship holds regardless of whether a given level of radioactivity has been reached after a short period of uptake or after a long period of uptake followed by washing.Although the action of exogenous cytokinins on excised plant organs is well documented (6,8,9) much less is known about their absorption. Studies on cytokinin uptake have been done with cultured cells (3) and with moss protonemata (5), but comparatively little work has been done with whole organs from higher plants (2, 10). Some interesting data about uptake of BA in Amaranthus half-seedlings have appeared in a recent report (4), but they were not discussed because cytokinin absorption was not the main issue of the paper.Earlier work done in our laboratory showed that treatments with lO-' M BA for as short as 1 min are sufficient to evoke longlasting responses in watermelon cotyledons (8). These results suggested that cotyledons are able to take up BA quite efficiently from the external medium. In this paper we present some characteristics of BA uptake and release in isolated cotyledons and some data on the relationship between total amount of BA taken up and magnitude of the hormonal response.MATERIALS AND METHODS Plant Material. Watermelon (Citrullus vulgaris Schrad.) seeds were bought from SRS seeds, Modesto, CA. The cotyledons were excised from the embryonic axis after 24 h of imbibition at 27 C in the dark (day zero) and immediately used for the uptake tests.Uptake of Labeled BA. Batches of 20 cotyledons were placed in a small Petri dish (50 mm diameter) containing one filter-paper disc imbibed with 1 ml 8-[4C]BA solution (1.34,Ci/,tmol) at l-4 M concentration. After an appropriate time of incubation in darkness at 27 C or 2 C the cotyledons were blotted dry, thoroughly rinsed with distilled H20 as described (8) and extracted in boiling 80%1o ethanol (v/v) for 1 h. For each uptake experiment we used 4 batches of 5 cotyledons that were extracted separately with 5 ml ethanol. One ml of the ethanol used for extraction was added to 10 ml Luma Gel in a scintillation vial. The radioactivity was determined in a Packard Tri-Carb scintillation counter with 78% efficiency. SD between replicate samples did not...
Some characteristics of uptake of 18-14CIN"-benzyladenine (BA) by watermelon (Citdus vulgaris Schrad., cv. Fairfax) cotyledons that were either excised immediately after 24 hours inhibition (day 0) or cultured in the dark for 48 hours on moist filter paper (day 2) have been compared.The uptake of BA seems to be passive in cotyledons of both kinds. The initial rate of uptake is, however, much slower In day 2 cotyledons. This is probably due to a higher resistance of cell membranes to BA influx. When the day 2 cotyledons are frozen and thawed, so that the membrane barrier is abolished, the amount of BA taken up is the same as in day 0 cotyledons.In spite of the lower rate of uptake, the physiological effects of BA in day 2 cotyledons are as strong as in day 0 cotyledons and occur with a shorter lag time. Sensitivity to BA seems indeed to start 24 to 48 hours after excision.Exogenous cytokinins accelerate development of excised cotyledons (2,4,7,15). We have shown (6) that continuous exposure to BA is not required for maximal effect. A treatment of 1 to 4 h with 10-' M BA is optimal, whereas a treatment of a few minutes suffices for a significant response. We used, for these experiments, cotyledons excised from the embryo after 24 h of imbibition (day 0). In these cotyledons, the responses to BA become apparent after 24 to 48 h lag time. In the present study, we observed that the lag can be greatly shortened if BA is supplied to cotyledons that have been grown in water for 2 d after excision.In a former paper (3), we described the main characteristics of BA uptake in cotyledons on day 0. The much prompter response of day 2 cotyledons suggested that BA uptake could be more efficient at this stage of development. In the present paper, we compare the characteristics of uptake by the two kinds of cotyledons.MATERIALS AND METHODS Watermelon (Citrullus vulgaris Schrad., cv. Fairfax) seeds were bought from SRS Seeds, Modesto, CA. The cotyledons were excised from the embryonic axis after 24 h of imbibition in distilled H20 at 27°C (day 0). They were then grown for 48 h more on moist filter paper in Petri dishes at 27°C in the dark.These day 2 cotyledons were used for the uptake tests.
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