Surfactant-Enhanced Penetration of Benzyladenine through Isolated Tomato Fruit Cuticular Membranes Peter D. Petracek, † Royal G. Fader, Moritz Knoche, ‡ and Martin J. Bukovac* Department of Horticulture, Michigan State University, East Lansing, Michigan 48824-1325 The effect of Triton X-100 (TX-100) on the penetration of benzyladenine (BA) through isolated tomato (Lycopersicon esculentum Mill.) fruit cuticular membranes was studied using finite and infinite dose diffusion and sorption/desorption systems. Finite dose penetration of BA (18 and 160 μM, pH 2 and 6) from donor droplets (3 μL) was characterized by an initial time lag, a maximum rate of penetration, and total penetration. TX-100 (0.1% w/v) increased maximum BA penetration rates 1.5-40-fold (pH 6, 18 and 160 μM) and total penetration 1.5-3-fold (pH 2 and 6, 18 and 160 μM BA). In infinite dose studies, rates of BA penetration (20 μM, pH 6.0) were increased ∼2-fold by TX-100. Surfactant effects were greatest when the surfactant was presented to the outer morphological surface of the cuticle (both native and dewaxed) regardless of whether TX-100 was penetrating in the same or opposite direction to BA. Sorption/desorption of BA was not affected by TX-100. Keywords: Cuticle; cytokinin; diffusion; Triton X-100; sorption; Lycopersicon esculentum INTRODUCTION Surfactants are used in spray solutions to enhance the performance of foliar-applied chemicals. This en- hancement has been attributed to (1) increased active ingredient (ai) solubility or dispersion in the spray solution, (2) improved wetting of the leaf surface, (3) modified physicochemical characteristics of the spray solution, and (4) enhanced cuticular penetration. Be- cause the cuticle is the rate-limiting step for foliar uptake, studies of isolated cuticle/penetrant interactions have been the focus of numerous investigations [for reviews see Bukovac and Petracek (1993), Bukovac et al. (1990), and Scho ¨nherr and Riederer (1989)]. The effect of an additive on ai penetration of the cuticle may be assessed, in part, by using infinite dose diffusion systems. Recent studies established that a nonionic surfactant, Triton X-100 (TX-100), increased steady-state cuticular permeation of the growth regula- tor naphthaleneacetic acid (NAA) and that the surfac- tant appeared to modify permeability of the cuticular waxes (Knoche and Bukovac, 1993a,b; Knoche and Bukovac, 1994). This observation was supported by studies on Triton X-45-enhanced penetration of NAA (Petracek et al., 1993). In this study, surfactant-enhanced penetration of benzyladenine (BA) through isolated tomato fruit Ly- copersicon esculentum Mill. cuticle was examined both in the presence and in the absence of epicuticular and cuticular waxes. A finite dose diffusion system (Buko- vac and Petracek, 1993) was used to determine the effects of surfactant on BA penetration from simulated spray droplet deposits, whereas the mechanisms of surfactant-enhanced penetration were then examined in infinite dose diffusion (Knoche and Bukovac, 1994) and sorption (Bukovac and Petracek, 1993) systems. MATERIALS AND METHODS Cuticle Isolation. Disks (17 mm diameter) were excised from mature tomato (L. esculentum Mill. cv. Pik Red) fruit grown without pesticide application. The cuticular membranes (CM) were enzymatically isolated (Orgell, 1955; Yamada et al., 1964), rinsed in deionized water, air-dried, and stored at room temperature. Dewaxed CM (DCM) were prepared by batch extracting CM with solvent (chloroform/methanol 1:1 v/v) 10 times over a 7-day period at 45 °C. Chemicals. Solutions of [ 14 C]BA [N-(phenylmethyl)-1H- purin-6-amine, specific activity ) 2.04 GBq mmol -1 , radio- chemical purity 97%] were prepared in 20 mM citrate buffer containing 1 mM NaN3 to prevent microbial growth. NaN3 was present in all citrate buffer solutions. For source of BA, see the Acknowledgment. The pH was adjusted to 2.0 and 6.0 using HCl and NaOH, respectively. Calculated ratios of cationic/nonionic BA were 19:1 for pH 2.0 and 1:500 for pH 6.0 as determined by the Henderson-Hasselbalch equation (Segel, 1976) and using 3.3 for the pK a of BA (Shafer, 1990). Therefore, BA was primarily cationic at pH 2.0 and nonionic at pH 6.0 (Figure 1). Surfactant solutions of 0.1% w/v (∼1.59 mM) TX-100 [R-[4-(1,1,3,3-tetramethylbutyl)phenyl]-ω-hy- droxypoly(oxy-1,2-ethanediyl)], a commercial polydisperse prepa- ration by Rohm and Haas (Philadelphia, PA; critical micelle concentration, cmc, 0.019% w/v), were prepared in 20 mM citrate buffer. Experimental Procedure. Finite Dose Diffusion. BA penetration from a droplet/deposit was measured using a system previously described (Bukovac and Petracek, 1993; Figure 2A). Isolated CM disks were weighed and checked for leaks by mounting (with vacuum grease) the CM disks in holders (plexiglass washers with 10 mm i.d.), sealing the mounted CM between two glass half-cells of the infinite dose diffusion apparatus (Figure 3A) with vacuum grease, and applying a slight hydrostatic pressure (∼3 cm of water) for 24 h. The mounted CM disks were transferred to finite dose diffusion units and sealed with vacuum grease. The wax-rich, * Author to whom correspondence should be addressed (fax 517/353-0890). † Present address: Florida Department of Citrus, CREC, Lake Alfred, FL 33850. ‡ Present address: Institute for Agronomy and Crop Science, Department of Horticulture, Martin-Luther-University of Halle-Wittenberg, 06099 Halle (Saale), Germany. 2346 J. Agric. Food Chem. 1998, 46, 2346-2352 S0021-8561(97)01093-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/28/1998