J. Pesticide Sci. 25, 144-146 (2000) Note An Efficient Method to Identify Oxyfluorfen Resistant Rice Lines by Seed Germination and Mortality of Seedlings Cheol Soo KIM, Hee Jae LEE, Oksoo HAN,* Kyoungwhan BACK, Eunyoung CHOI, Ja Ock GUH, Youn Tae CHI and Do Jin LEEt Institute of Biotechnology, Institute of Agricultural Science and Technology, College of Agriculture, Chonnam National Univer- sity, 300 Yongbong-dong, Buk-gu, Kwangju 500-757, Korea tDepartment of Agricultural Education, Sunchon National University, 315 Maegok-dong, Sunchon 540- 742, Korea (Received September 3, 1999; Accepted December 27, 1999) Key words: herbicide resistance,oxyfluorfen,protoporphyr- inogen oxidase, rice. INTRODUCTION Oxyfluorfen [2-chloro-l-(3-ethoxy-4-nitrophenoxy)-4-(tri- fluoromethyl)benzene]and structurally related Biphenyl ether compounds exert their herbicidal effect by inhibiting protoporphyrinogen oxidase (Protox, EC 1. 3. 3. 4), which is located in the plastid envelope. 1'2 The competitive inhibi- tion of Protox eventually leads to the massive accumulation of photosensitizing protoporphyrin IX, which causes mem- brane lipid peroxidation and ultimately cellular death in the presence of molecular oxygen and light.3, 4) Since Protox from Bacillus subtilis is poorly inhibited by diphenyl ether herbicides, 5, 6 the developmentof transgenic plants resistantto these herbicides is plausible. In fact, we have successfully generated transgenic tobacco plants resistant to oxyfluorfen via expression of the B. subtilis Protox gene in tobacco plants' and are trying to expand this technique to other plants such as rice and turfgrass. During the course of developing transgenic rice plants resistant to oxyfluorfen, an efficient screening method was needed to differentiate resistant transgenic lines from sensitive lines at an early stage of growth such as the germination and/ or seedling stage. The currently available biochemical method is a leaf disc assay, 89 involving the estimation of electrolyte leakage by measuring conductivity change, quantitation of lipid peroxidation by measuring malondial- dehyde (MDA) production, or the determination of chloro- phyll content. However, these methods require destructive sampling and the parameters determined by these methods largely depend on the growth stage of individual lines and thus, they are applicable only when individual plants reach a certain stage of growth. In an effort to develop an efficient screening method, seeds from transgenic rice plants at T1 generation were treated with oxyfluorfen and the rate of seed germination and the mortality of the emerged seedlings were determined and compared with the degree of lipid peroxidation determined by MDA produc- tion. MATERIALS AND METHODS 1. Plant Materials The transgenic rice plant (Oryza sativa cv. Nakdong) containing the B. subtilis Protox gene with a ubiquitin pro- moter in the pGA 1611 vector was produced by the previously reported Agrobacterium-mediated method and will be de- scribed in detail elsewhere. 2. Seed Germination and Mortality of Seedlings The rice seeds peeled were soaked in distilled water, and kept at 4C for 1 day in darkness. These seeds (10 of nontransgenic seeds and 9-10 of transgenic seeds for each concentration of oxyfluorfen) were incubated at 4C for 1 day in darkness in the presence of oxyfluorfen at concentrations from 0.01 to 1000jM in 1%(v/v) acetone. The oxyfluorfen- treated seeds were germinated on two sheets of wet filter paper at 30C in darkness. After the dark incubation for 2 days, seed germination rate was recorded. The seed was consid- ered to have germinated when the length of plumule exceeded 2 mm. Only germinated seeds were transferred to l0-cm diameter plastic pots containing a commercial soil substrate, placed in a greenhouse, and grown at 30±5/20+5C, day/ night temperature with an approximately 14-hr photoperiod. The mortality of the seedlings was determined by visual rating with complete seedling death or no new growth from the seedlings and their shoot fresh weights were determined 3 days after transplanting. 3. Leaf Disc Assay In separate experiments, the nontransgenic and the trans- genic rice seeds sprouted by soaking in a fungicidal prochlor- az solution for 1 day and subsequently in water for 2 days were grown in the same greenhouse as above until they had two true leaves. Tissues from the first and second true leaves were treated with oxyfluorfen as before9 by cutting 4-mm leaf squares (0.2 g fresh weight) with a razor blade, and then placing them in a 6-cm diameter polystyrene petri dish containing 5 ml of 1 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.5) with or without the herbicide dissolved in acetone. Control contained the same amount of the solvent without the herbicide. The final concentration of acetone in all dishes was 1% (v/v). The tissues were incubated in a growth chamber at 25C in darkness and then exposed to * To whom correspondence should be addressed.