Abstract：Targeted mutagenesis is becoming an important way to modify genes in various cells and organisms, and has been widely used for functional study of genes and for genetic modification in plants and animals. Currently, three technologies, including zinc-finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN)， and clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9), have been developed for targeted mutagenesis. Among them, TALEN technology has been successful in targeted gene modification in many agricultural plants, and has become an effective tool for molecular breeding in crops. In this study, we employed TALEN to generate site-specific modification in CYP81A6, which confers bentazone resistance in rice (Oryza sativa), to obtain bentazone-sensitive mutant applicable in rice hybrid breeding. TALEN-targeted site was selected near the 5' end of CYP81A6 coding region to ensure complete knockout of gene function. A total of 110 T0 transgenic plants were generated by Agrobacterium-mediated transformation of rice callus (Japonica, Wuyunjing 7). High resolution melting (HRM) analysis indicated that 3 T0 transgenic lines carried targeted mutation (i.e. mutagenesis frequency of 2.73%). These 3 lines were sensitive to bentazone in the field test. Sequence analysis revealed multiple mutations in CYP81A6 in the three lines, indicating the transgenic lines as chimera. The 3 T0 transgenic lines were self-pollinated, and the T1 population were subjected to bentazone-sensitivity test. We found many T1 transgenic seedlings still sensitive to bentazone, but none was homozygous mutant. T1 non-transgenic seedlings were all resistant to bentazone-treatment. These results indicated that the TALEN employed in this study was not efficient in generating mutations in germline cells, although it was active in somatic cells. The wide presence of chimeric mutants in T0 and T1 plants indicated that mutations generated by TALEN need to be reconfirmed in the next generation, preferably in the plants that does not contain the transgene. Although an inheritable mutant was not obtained, the study generated valuable information that can improve future experiments in targeted mutagenesis and screening of mutant plants.
