Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Page 31 Page 32 Page 33 Page 34 Page 35 Page 36 Page 37 Page 38 Page 39 Page 40 Page 41 Page 42 Page 43 Page 44 Page 45 Page 46 Page 47 Page 48 Page 49 Page 50 Page 51 Page 52 Page 53 Page 54 Page 55 Page 56 Page 57 Page 58 Page 59 Page 60 Page 61 Page 62 Page 63 Page 64 Page 65 Page 66 Page 67 Page 68 Page 69 Page 70 Page 71 Page 72 Page 73 Page 74 Page 75 Page 76 Page 77 Page 78 Page 79 Page 80 Page 81 Page 82 Page 83 Page 84 Page 85 Page 86 Page 87 Page 88 Page 89 Page 90 Page 91 Page 92 Page 93 Page 94 Page 95 Page 96 Page 97 Page 98 Page 99 Page 100 Page 101 Page 102 Page 103 Page 104 Page 105 Page 106 Page 107 Page 108 Page 109 Page 110 Page 111 Page 112 Page 113 Page 114 Page 115 Page 11671 Advancing Seed in Alberta | fall.2016 The SSN-2 technique uses a donor DNA, which is a copy of the target DNA region with a small modification. During repair, the plant will use this template for the repair, and the small modi- fication will be introduced into the plant’s genome (targeted mutation). The repair template of the SSN-3 application 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 meganu- cleases 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, similar results can be obtained with SSN-1, SSN-2 and clas- sical mutagenesis methods, with one big difference—classical mutagenesis leads to thousands of random mutations, whereas SSN-1 and SSN-2 lead to single specific mutations in a targeted gene. Another disadvantage of classical mutagenesis methods is this necessitates a selection for plants with the intended mutations, 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 identical to the plant’s genetic material—except 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 species without crossing any species barrier. It is this creation of addi- tional genetic variation that is absolutely crucial and fundamental to plant breeding. RNA-Dependent DNA methylation RNA-dependent DNA methylation (RdDM) relies on the plant’s 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 foreign origin and methylates the matching DNA, ultimately blocking the expression of the gene (Figure 3). Figure 2. Simplified illustration of ODM. The left DNA helix (light blue/red) with oligonucleotide template (tan/red) 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. Illustration courtesy of NBT Platform viral gene + viral gene viral gene gene RNA degradation fragments RISC RISC forms complex with RNA RISC-RNA finds matching DNA ... and attaches methyl (CH3)- groups to DNA ... which blocks activity of the gene natural target gene natural target gene CH3 CH3 CH3 CH3 CH3 CH3 recombinant gene + CH3 CH3 CH3 CH3 CH3 CH3 RNA-Dependent DNA Methylation Reverse Breeding It is not possible to exactly reproduce a heterozygous plant via seeds. Only vegetative reproduction will allow for an exact copy. However, seed companies are geared to reproduce and commer- cialise their elite plant varieties by means of seeds, as vegetative reproduction is often too expensive, technically cumbersome, and commercialization is often logistically impossible. Reverse breeding uses a genetic modification step to suppress the recom- bination of chromosomes, followed by specific tissue culture to create homozygous parent lines. These lines are then used to stably produce the heterozygous elite plants through seed (Figure 4). GM Rootstock Grafting With this technique, the top part of a plant, called the scion, is grafted onto a GM rootstock (Figure 5). The resulting combined Figure 3. Simplified graphical representation of RdDM. On the left side, the plant’s 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 Illustration courtesy of Wageningen UR