An efficient method to identify oxyfluorfen resistant rice lines by seed germination and mortality of seedlings

「INTRODUCTION」 Oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene] and structurally related diphenyl 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 inhibition of Protox eventually leads to the massive accumulation of photosensitizing protoporphyrin IX, which causes membrane 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 development of transgenic plants resistant to 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 plants7) 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, 8, 9) involving the estimation of electrolyte leakage by measuring conductivity change, quantitation of lipid peroxidation by measuring malondialdehyde (MDA) production, or the determination of chlorophyll 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 production.