Metabolism of 2,4-Dichlorophenoxyacetic Acid in 2,4-Dichlorophenoxyacetic Acid-Resistant Soybean Callus Tissue

Abstract

MATERIALS AND METHODS Three 2,4-dichlorophenoxyacetic acid (2,4-D)-resistant root callus tissue lines of Glycine max L. MerriH var. Acme were derived by culturing callus tissue 2 to 6 months on 40 milligrams per liter 2,4-D and designated 40R, 40B, and 40C. Tissue line 40R had a lower level of 2,4-D uptake in 2-week-old tissue which disappeared in 3.5-week-old tissue and less free 2,4-D following incubation for 24 hours with I1-'4C12,4-D. This tissue line accumulated more hydroxylated glycosides of 2,4-D than did nonresistant tissue. Tissue line 40B showed no difference in uptake of 2,4-D when compared to nonresistant tissue but it did contain less free 2,4-D and more hydroxylated glycosides. The metabolism of 2,4-D in the 40C tissue line did not differ significantly from nonresistant tissue although uptake was less. The 40R line reverted to the same 2,4-D sensitivity as Acme root caDlus following six transfers on 10 micromolar naphthaleneacetic acid medium. The altered 2,4-D uptake and metabolism characteristic of 40R were also lost. The levels of amino acid conjugates of 2,4-D in the resistant root callus tissue lines were either lower or not significantly different from the Acme tissue lines. Therefore, variations in uptake and metabolism of 2,4-D do not wholly explain the resistance of the derived tissue Unes, and perhaps modification of the active site or compartmentation is involved. Species-specific tolerance of plants to herbicides is well recognized. Grasses are generally resistant to phenoxy herbicides while broadleaf plants are susceptible. A basis for species-specific plant resistance to 2,4-D has been examined in tissue cultures. In monocots, the primary metabolites found are glycoside esters (6, 15) or glycosides of hydroxylated metabolites (1, 5). Susceptible dicots such as soybean, sunflower, and tobacco also possess these same detoxification mechanisms but to a lesser degree (4, 5). These plants primarily conjugate 2,4-D with amino acids, and the conjugates are biologically active (7). Evidence exists that the hydrox-ylated glycosides are inactive (9, 11). There are numerous examples of selection for 2,4-D resistance or differential tolerance in tissue culture: Citrus sinensis, Daucus carota, Lotus corniculatus, Nicotiana sylvestris, and Trifolium repens (8, 10, 14, 16-18). Despite the variety of examples, the metabolism of 2,4-D in resistant or differentially tolerant tissue has received little attention. We have isolated several soybean root callus tissue lines capable of growing on an elevated 2,4-D concentration and examined the metabolism of 2,4-D in three of these tissue lines. Plant Material and Growing Conditions. Soybean seeds (Glycine max [L.] Merrill var. Acme) obtained from the United States Department of Agriculture soybean germplasm collection were surface sterilized in 2% NaOCI, rinsed in sterile distilled H20, and allowed to germinate on moistened sterile filter paper. Sterile roots were placed on agar-solidified modified Miller's Medium (12) containing sucrose (3%), NAA2 (10 ELM), and kinetin (2.32 ,UM) under continuous low-intensity fluorescent light (0.5 ,uE/m2. s) at 25°C. After callus had been initiated, the tissue was carried through seven transfers (one transfer per month). Then some of the callus was placed on media containing 40 mg/l (181 ,tM) 2,4-D instead of NAA. After several (2-6) months on the 2,4-D media, three lines of 2,4-D-resistant callus tissue was isolated. The tissue lines are designated 40R, 40B, 40C. The stock cultures of Acme callus grown continuously on NAA media served as the control tissue. Four Acme and 40R callus tissue pieces (-30 mg per piece) were transferred to 50 ml of Miller's agar medium in 125-ml flasks containing no auxin or various concentrations of NAA, 2,4-D, or 2,4,5-T as indicated. Each treatment had five replicates, and the tissue was weighed after 1 month. Prior to metabolism studies, all 2,4-D-tolerant tissue was grown for 2 to 4 weeks on media containing 10 ,UM NAA. Two-to 4-week-old callus tissue (0.9-6.8 g) was aseptically transferred to 125-ml flasks containing 20 or 50 ml liquid medium (without added auxin) to which 4.3 to 113.2 !LM [l-4C]2,4-D (10.1 mCi/ mmol, ICN) was added. Tissues were incubated for 24 h on a shaker, and then rinsed with sterile H20 and weighed. The tissue was ground in a VirTis 45 tissue homogenizer with 10 times its weight of hot 95% ethanol. The homogenate was filtered and the residue washed with 80%o ethanol. The filtrate was concentrated by rotary evaporation (40°C), adjusted to pH 3 (H3P04), and extracted four times with equal volumes of diethyl ether. The aqueous phase was extracted three times with equal volumes of 1-butanol saturated with H20. The butanol fraction was concentrated in a rotating evaporator at 50°C to 0.5 ml. This fraction was transferred to a flask and evaporated to dryness under a jet of N2. Twenty ml of distilled H20 was added and the pH adjusted to 5 (NaOH) prior to incubation with 18-glucosidase (7 mg of Emulsin, United States Biochemical Corp.) for 6 h on a shaker at 27°C. The solution then was acidified to pH 3 (H3PO4) and extracted with diethyl ether. The ether fractions were chromatographed on Supelcosil 12A thin-layer plates containing a fluorescent indicator (zinc silicate) or Silica gel 60 F2M plates (EM Reagents, MC/B Manufacturing Chemists, Inc.). Metabolites were located by autoradiography and 2 Abbreviations: NAA, naphthaleneacetic acid; 2,4,5-T, 2,4,5-trichloro-phenoxyacetic acid. 104