REVIEW Progress of targeted genome modification approaches in higher plants Teodoro Cardi 1 • C. Neal Stewart Jr. 2 Received: 11 February 2016 / Accepted: 21 March 2016 / Published online: 29 March 2016 Ó Springer-Verlag Berlin Heidelberg 2016 Abstract Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative conse- quences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recom- bination for gene integration commenced soon after the first demonstration that transgenic plants could be pro- duced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chi- meric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial devel- opment and implementation in plant breeding will be affected by governmental regulations. Keywords Homologous recombination Á Recombinases Á Oligonucleotide-directed mutagenesis Á Nucleases Á Targeted mutagenesis Á Site-specific integration Introduction In 1983, four independent studies were published on the stable genetic transformation of higher plants (Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; Murai et al. 1983). In light of conventional breeding technologies available then, these papers represented a great leap for- ward in genetic modification technology. The ability to choose specific genes in plant genomes to modify for cultivar development revolutionized crop breeding. Sub- sequently, the general concept was refined to control transgene expression in space and time; i.e., in specific tissues, developmental stages and environments. Not only could new heterologous genes be introduced and expressed in the plant chassis, but endogenous gene expression could be downregulated using antisense or RNAi technologies (Koch and Kogel 2014; Sheehy et al. 1988). Moreover, transgenes could also be integrated into plastid genome (Svab et al. 1990). More recently, the use of plant sequences derived from cross-compatible species was advocated in the so-called ‘‘cisgenic’’ gene transfer, with Communicated by M. Mahfouz. & Teodoro Cardi teodoro.cardi@crea.gov.it 1 Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria (CREA), Centro di Ricerca per l’Orticoltura, Via Cavalleggeri 25, 84098 Pontecagnano, Italy 2 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA 123 Plant Cell Rep (2016) 35:1401–1416 DOI 10.1007/s00299-016-1975-1