The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here we describe methods for the targeted modification of plant genomes using transcription activator like effector nucleases (TALENs). Methods were optimized using Nicotiana tabacum (tobacco) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing (SSA) assay in which TALEN cleavage creates a functional YFP gene, enabling quantification of TALEN activity by flow cytometry. SSA activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high throughput DNA sequencing of PCR products encompassing the TALEN recognition sites. TALENs made with optimized scaffolds introduced targeted mutations in ALS in 30% of transformed cells, and frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 31.5% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322 bp donor molecule differing by 6 bp from the ALS coding sequence, 3.5% showed evidence of targeted gene replacement. The optimized reagents described and implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.