Feasibility of new breeding techniques for organic farming Martin Marchman Andersen, Xavier Landes, Wen Xiang, Artem Anyshchenko, Janus Falhof, Jeppe Thulin Østerberg, Lene Irene Olsen, Anna Kristina Edenbrandt, Suzanne Elizabeth Vedel, Bo Jellesmark Thorsen, Peter Sandøe, Christian Gamborg, Klemens Kappel, Michael G. Palmgren  Trends in Plant Science  Volume 20, Issue 7, Pages 426-434 (July 2015) DOI: 10.1016/j.tplants.2015.04.011 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Breeding techniques available for rewilding of crop plants and their legal feasibilities in the European Union (EU). (A) Introgression breeding is the standard method used to introduce genes and traits from wild plants into domesticated crops. This method uses an initial cross between the crop and the wild relative of interest followed by repeated backcrossing to the domesticated crop to erase as much genetic material from the wild relative as possible while keeping the trait of interest. Molecular markers can be used to track the trait of interest through the crosses, a process called ‘marker-assisted breeding’. However, introgression breeding is time consuming and technically challenging when more than one gene is being selected for, and it is often difficult to get rid of closely linked undesired genes. Given that introgression breeding does not involve genetically modified (GM) techniques, the product is not classified as a genetically modified organism (GMO) in the EU. (B) Transgenesis allows for the transfer of a desired gene from an unrelated organism into the domesticated crop. The process is based on the random genomic insertion into the crop plant of genetic material by the soil bacterium Agrobacterium tumefaciens. Typically, all of the inserted elements are transgenic. The gene in question is cloned into a binary vector system along with a selection marker and between short transfer DNA (T-DNA) border sequences of bacterial origin. Subsequently, Agrobacterium inserts gene material between T-DNA border sequences into the crop genome. T-DNA insertion by Agrobacterium occurs widely in nature and the major crop sweet potato (Ipomoea batatas) was recently identified as naturally transgenic because it harbors T-DNA [92]. (C) Cisgenesis allows for the direct transfer of complete genes from wild relatives into domesticated crops. Given that the transfer process can be restricted to a single gene only, the method avoids co-transfer of genetically linked genes that could have undesired effects. The genes introduced by cisgenesis can be in their natural configuration and contain their own promoter, regulatory elements, and introns. Given that DNA is typically introduced by Agrobacterium-mediated transformation, transgenic material is introduced concomitant with the gene of interest. However, selection markers that originate from rice and other plants are now available, and it is possible to remove the marker genes after they have served their purpose. Furthermore, T-DNA sequences can be modified so that they are identical to sequences found in the plant (in which case they are known as P-DNAs). Given that cisgenesis involves the use of several GM techniques, the product would have to be classified as a GMO in the EU. (D) Precision breeding,; also called ‘genome editing’,; is a new technique in plant breeding that offers even greater precision than cisgenesis [93–96]. This technique can be used to create specific mutations and has the potential to generate plants that differ from the original plant at a single nucleotide position and otherwise carry no signs of the modification. Precision breeding can be used when the wild type gene differs from the gene in the domesticated crop at a single nucleotide. Several variants of the technique exist, such as those involving zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system [96]. Generally, a system that has been engineered to cleave a specific DNA sequence is introduced into the nuclei of plant cells, where it creates a DNA double-strand break at the exact position of the recognized DNA sequence. The break is subsequently repaired by the DNA repair machinery of the plant cell. This process can be imprecise (by the nonhomologous end-joining pathway) or precise (by the homologous recombination pathway) [96,97]. In the second case, targeted specific mutations or insertions can be introduced. Given that precision breeding techniques are so new, there are still no examples of crops rewilded by this technique. Precision breeding involves the use of a GM technique, which implies that the plant product could be classified as a GMO. However, this is still uncertain in the EU. Trends in Plant Science 2015 20, 426-434DOI: (10.1016/j.tplants.2015.04.011) Copyright © 2015 Elsevier Ltd Terms and Conditions