Table 2.
Plant breeding techniques comparison according to criteria defining their status under Directive 2001/18/EC in the light of the Judgment.
| Breeding Technique | History of use in plant breeding | Annex I A Part 1 Directive 2001/18/EC? | Annex I A Part 2 Directive 2001/18/EC? | “[M]utagenesis” or “cell fusion [ … ]”? | “[I]nvolve [ … ] recombinant nucleic acid molecules” | “[U]se” lato sensu of recNA (introduction in the plant of DNA or RNA sequences)? | “[U]se” stricto sensu of recNA (stable insertion of “heritable” DNA sequences)? | Classification according to Directive 2001/18/EC | Out of the scope of Directive 2001/18/EC? | Potentially exemptible? |
|---|---|---|---|---|---|---|---|---|---|---|
| Crossing and selection (classical breeding) | Since 1870's (Mendel, 1901) | No | Yes | No | No | No | No | Art 2(2) + Annex I A Part 2 | Yes | – |
| In vitro fertilization | Since 1930's (Sharma et al., 1996) | No | Yes | No | No | No | No | Art 2(2)(b) + Annex I A Part 2 | Yes | – |
| Polyploidy induction | Since 1940's (Blakeslee and Avery, 1937) | No | Yes | No | No | No | No | Art 2(2)(b) + Annex I A Part 2 | Yes | – |
| Random mutagenesis (chemicals, radiations) | Since 1930's (Stadler, 1928) | yes (but exempted) | No | Yes (“mutagenesis”) | No | No | No | Art 3(1) + Annex I B | Yes (exempted GMO) | – |
| Protoplasts fusion between sexually compatible species | Since 1970's (Carlson et al., 1972) | yes (but exempted) | No | Yes (“cell fusion [ … ]”) | No | No | No | Art 3(1) + Annex I B | Yes (exempted GMO) | – |
| Protoplasts fusion between sexually incompatible species | Since 1970's (Carlson et al., 1972) | yes (because not in the other annexes) | No | No | No | No | No | Art 2(2)(a) + Annex I A Part 1 | No | No |
| “Classical” transgenesis | Since 1980's (Zambryski et al., 1983) | yes (expressly mentioned) | No | No | Yes | Yes | Yes | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Microinjection/macroinjection and microencapsulation | Since 1980's (Reich et al., 1986) | yes (expressly mentioned) | No | No | Yes a | Yes | Yes/No b | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Agro-infiltration | Since 1990's (Grimsley et al., 1986) | yes (not in the other annexes) | No | No | Yes | Yes | No | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Oligonucleotide-Directed Mutagenesis (ODM) | Since 2000's (Zhu et al., 1999) | yes (recital 17 not ascertained) | No | Yes (“mutagenesis”) | Yes/No* c | Yes/Noc | No | Art 2(2)(a) + Annex I A Part 1 | No | Yes* |
| Intragenesis | Since 2000's (Rommens et al., 2007) | yes (not in the other annexes) | No | No | Yes | Yes | Yes | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Cisgenesis | Since 2000's (Schouten et al., 2006) | yes (not in the other annexes) | No | No | No* (if no T-DNA) | No (if no T-DNA) | No (If no T-DNA) | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Transgrafting (GM scion on non-GM rootstock, or vice-versa) | Since 2000's (Lifschitz et al., 2006) | yes (not in the other annexes) | No | No | Yes | Yes/No (depends on the part harvested) | Yes/No (depends on the part harvested) | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Reverse breeding | Since 2010's (Dirks et al., 2009) | yes (not in the other annexes) | No | No | Yes | No (in the final product) | No (in the final product) | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Gene editing: Targeted mutagenesis using site-directed nucleases (SDN1) without insertion of the nuclease gene (transient transformation, RNA, RNP (ribo-nucleo protein), null segregant) | Since 2010's (Jiang et al., 2013; Woo et al., 2015) | yes (recital 17 not ascertained) | No | Yes (“mutagenesis”) | Yes/No* c | Yes/No c | Yes/No c | Art 2(2)(a) + Annex I A Part 1 | No | Yes* |
| Gene editing: Targeted mutagenesis using site-directed nucleases (SDN1) with insertion of the nuclease gene | Since 2010's (Shan et al., 2013) | yes (not in the other annexes) | No | No | Yes | Yes | Yes | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Gene editing: Allele swap using site-directed nucleases (SDN2) without insertion of the nuclease gene (transient transformation, RNA, RNP (ribo-nucleo protein), null segregant) | Since 2010's (Shi et al., 2017) | yes (recital 17 not ascertained) | No | Yes (“mutagenesis”) | Yes/No* c | Yes/No c | Yes/No c | Art 2(2)(a) + Annex I A Part 1 | No | Yes* |
| Gene editing: Allele swap using site-directed nucleases (SDN2) with insertion of the nuclease gene | Since 2010's (Shi et al., 2017) | yes (not in the other annexes) | No | No | Yes | Yes | Yes | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Gene editing: Targeted transgenesis using site-directed nucleases (SDN3) | Since 2010's (Li et al., 2013) | yes (not in the other annexes) | No | No | Yes | Yes | Yes | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Gene regulation using site-directed effectors (activators/repressors /epigenetic factors) | Since 2010's (Piatek et al., 2015) | yes (not in the other annexes) | No | No | Yes | Yes | Yes/Nod | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Gene regulation using site-directed nucleases targeting RNAs (SDN4) (stable integration) | Being developed (Shmakov et al., 2015) | yes (not in the other annexes) | No | No | Yes | Yes | Yes | Art 2(2)(a) + Annex I A Part 1 | No | No |
| RNA dependent DNA methylation (RdDM) | Being developed (Ruiz-Ferrer and Voinnet, 2009) | yes (not in the other annexes) | No | No | Yes | Yes | Yes/Nod | Art 2(2)(a) + Annex I A Part 1 | No | No |
| Genome editing using site-directed recombinases | Being developed in animals, soon plants (Mercer et al., 2012) | yes (not in the other annexes) | No | No | Yes | Yes | Nod | Art 2(2)(a) + Annex I A Part 1 | No | No |
aInitially, these techniques were developed to transfer “recombinant nucleic acid molecules”.
bProvided that a stable insertion is not carried out.
c“No” provided that the allele sequence is already present in the species gene pool.
d“No” in case of transitory transformation or null segregant.
Nonexhaustive list of plant breeding techniques mentioned in Directive 2001/18/EC, in SAM (2017) and new approaches currently being developed, the descriptions of which can be found in the references mentioned inside the table.
All the techniques listed result in “an organism [ … ] in which the genetic material has been altered” (see question “1)” in Table 1 ).