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 tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS)
gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN
cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry.
Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous
targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN
recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted
gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or
enrichment regimes: 32% 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, 4% showed evidence of targeted gene replacement. The optimized reagents
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.