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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Selectable Tolerance to Herbicides by Mutated Acetolactate Synthase Genes Integrated into the Chloroplast Genome of Tobacco^1,[OA]

Laboratory of Plant Molecular Improvement and Global Center of Excellence Program, Graduate School of Nutritional and Environmental Science, University of Shizuoka, Shizuoka 422–8526, Japan (M.S., M.G., M.H., H. Kobayashi); Life Science Research Institute, Kumiai Chemical Industry Co., Ltd., Kikugawa, Shizuoka 439–0031, Japan (T.S., N.I.); Research Institute of Innovation Technology for the Earth, Kizugawa, Kyoto 619–0292, Japan (H. Kanamoto); Plant High Technology Institute, Takayama Science Plaza, Ikoma, Nara 630–0101, Japan (K.I.-T.); and Graduate School of Biological Science, Nara Institute of Science and Technology, Ikoma, Nara 634–0813, Japan (A.Y.)

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hirokazu Kobayashi (hirokoba{at}u-shizuoka-ken.ac.jp).

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.120519

^* Corresponding author; e-mail hirokoba{at}u-shizuoka-ken.ac.jp.

Received April 15, 2008; accepted May 20, 2008; published May 30, 2008.