Effect of Glyphosate on Ethylene Production in Tobacco Callus

Abstract

Glyphosate (N-phosphonomethylglycine) caused a significant decrease or a slight increase in ethylene production in tobacco callus (Nicotiana tabacum L.) depending on the concentration of indole-3-acetic acid (LIA) present in the medium. IAA stimulated ethylene production, but a pretreat-ment with glyphosate greatly reduced the IAA-induced ethylene production. Inasmuch as glyphosate treatment promoted the metabolism of IAA, the decrease in ethylene production induced by glyphosate is attributed to the rapid loss of free IAA in the treated tissue. Glyphosate (N-phosphonomethylglycine) is a promising, broad-spectrum herbicide. It also promotes sucrose accumulation in sugarcane (13) and maturity in grain sorghum (3). In searching for its mode of action in plants, Lee (8, 9) has found that tobacco callus pretreated with glyphosate had significantly less free IAA and higher rates of oxidation and conjugation of IAA than that in the control callus without glyphosate treatment. As IAA and other auxins are known to promote ethylene production in plants (1, 4, 10, 11, 14, 16) and the level of free IAA in plant tissues parallels ethylene production (5, 6), it is of interest to find out whether ethylene production is affected by glyphosate. In this work, we report that glyphosate significantly decreases the IAA-induced ethylene production in tobacco callus. MATERIALS AND METHODS Plant Material. The tobacco callus (Nicotiana tabacum L. cv White Gold) was grown on M-S medium (12) supplemented with 4 ,UM each of IAA and kinetin. The culture was grown in darkness at 27°C for 2 weeks, after which uniform pieces were used for experiments. The tobacco callus was chosen because it was sensitive to glyphosate in growth response (7) and IAA metabolism (8, 9). Glyphosate Treatment. Tobacco calluses were transferred to fresh M-S medium containing varied concentrations of IAA and glyphosate as indicated in the table and figures. They were kept in darkness at 27°C for 1 d. In some experiments, the calluses were grown with glyphosate for 1 d before being exposed to fresh IAA. Each treatment had six replicates and each experiment was repeated at least two times. Ethylene Measurement. After treatment with glyphosate and/ or IAA, each callus was removed from the medium, blotted on Kleenex tissue, and weighed. The fresh weight of each callus was about 1 g. Then the callus was transferred to an all-glass flask (40 ml) fitted with a 6-mm i.d. sampling port plugged with a silicone seal, which was the only area of exposure to non-glass material during ethylene collection. The flasks were kept in darkness at 27°C for 5 h. A 1-ml air sample was removed for analysis of ethylene. A Bendix 2300 GC equipped with a flame ionization detector and a nickel column (2.5 m x 3 mm i.d.) filled with Chromosorb 102 (80-100 mesh) was used. The column temperature was 60°C and the flow-rate of the N2 carrier gas was 30 ml/ min. The retention time for ethylene was 3.2 min and a full-scale pen deflection was given by 16 ng of ethylene on a 3380 Hewlett-Packard integrator. IAA Measurement. Extraction and separation of free IAA from the tobacco callus were carried out as previously described (8). The fraction containing IAA eluted from the Whatman Partisil-10-ODS column of the HPLC was concentrated and injected into the Whatman Partisil-10-SAX column (25 x 0.46 cm) with a mobile phase of 1% acetic acid at a flow of 1 ml/min and a pressure of 650 p.s.i. The amount of IAA was measured with a UV detector at 280 nm and a Shimadzu CRIB integrator using authentic IAA as the standard. The recovery of IAA was estimated by including a known amount of [2-14C]IAA in the sample at the beginning of extraction and by counting the radioactivity of [2-4C]IAA recovered at the end of isolation. RESULTS AND DISCUSSION Effect on IAA-Induced Ethylene Production. The tobacco callus produced ethylene but the amount of ethylene released varied with the age of the culture and the concentration of IAA. A 2-week-old callus, which had been grown with 4 tlM IAA at the beginning, produced about 20 ng of ethylene/g fresh weight.h. When such a callus was transferred to a fresh medium with 10