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EUROPEAN-SEED.COM I EUROPEAN SEED I 25 small modification will be introduced into the plants genome targeted mutation. The repair template of the SSN-3 applica- tion type contains a complete new gene. Using SSN-3 intragenes cisgenes see below or transgenes can be introduced gene addition. In any of the three ways described above with SSN a gene of interest can be mutated replaced or knocked out Figure 1. CRISPR- Cas9 zinc-finger nucleases ZFNs TALENs and meganucleases are all different variants of SSN. For decades plant breeders have been using classical mutagenesis methods such as chemicals or ionizing radiation. In a way sim- ilar results can be obtained with SSN-1 SSN-2 and classical mutagenesis methods with one big differenceclassical mutagenesis leads to thousands of random mutations whereas SSN-1 and SSN-2 lead to single specific muta- tions in a targeted gene. Another disadvantage of classical mutagenesis methods is this necessitates a selection for plants with the intended muta- tions but also plant breeders must carry out several generations of backcrossing to get rid of unwanted mutations. These two latter steps are much simpler and faster when using SSN-1 or SSN-2. OLIGONUCLEOTIDE- DIRECTED MUTAGENESIS The technique oligonucleotide-directed mutagenesis ODM uses oligonucleotides small molecules into which in a similar manner to SSN-2 a small repair template is introduced into the plant cell which is identi- cal to the plants genetic materialexcept for the desired change. After the DNA repair process plants are selected where the modification has been copied into the DNA. The difference with SSN-2 is no genetic construct is copied into the DNA of the plant itself. The small repair molecule that is used remains briefly in the plant cell and is quickly degraded Figure 2. This method only works in plants that can be regenerated from protoplasts. It is important to mention that with SSN-1 SSN-2 and ODM additional genetic variation is created within an existing spe- cies without crossing any species barrier. It is this creation of additional genetic variation that is absolutely crucial and fundamental to plant breeding. RNA-DEPENDENT DNA METHYLATION RNA-dependent DNA methylation RdDM relies on the plants defence system RNA- induced silencing complex RISC which is activated by small double-stranded RNA molecules from viruses for example. The system forms a complex with the RNA of for- eign origin and methylates the matching DNA ultimately blocking the expression of the gene Figure 3. REVERSE BREEDING It is not possible to exactly reproduce a heterozygous plant via seeds. Only vegetative reproduction will allow for an FIGURE 2. Simplified illustration of ODM. The left DNA helix light bluered with oligonucleotide template tanred containing one intended mismatch dark blue. After the endogenous DNA repair mechanism has copied the change pink into the DNA the template is degraded. The strands return to their original form not shown and the DNA repair mechanism copies the intended change of one strand into the complementary strand successfully completing the process. FIGURE 3. Simplified graphical representation of RdDM. On the left side the plants natural defence system leading to methylation of a viral gene. On the right side recombinant-derived RNA molecules guide the RISC to its natural counterpart resulting in DNA methylation and a subsequent blocking of gene activity. The recombinant gene contains fragments of the natural gene to be targeted. IllustrationcourtesyofNBTPlatformIllustrationcourtesyofWageningenUR