1. Plant Systems: Genetic Technologies and Constraints
Kent J. Bradford
Department of Plant Sciences
Seed Biotechnology Center
University of California, Davis

2. Plant Genetic Modification Technologies
Grafting – involves utilizing traits from two varieties/species by grafting.
Widely used in horticulture.
No genetic mingling assumed, but recently been shown to occur near graft site.
Generally limited to within a taxonomic genus or family.
Unregulated. Status if only root stock is GE is not yet determined.

3. Plant Genetic Modification Technologies
Sexual crosses – involves transfer of genes via sexual crossing and subsequent chromosomal segregation and recombination.
Can be used to introduce traits from wild species, if sexually compatible (or through embryo rescue, etc.).
Changes undirected (based on recombination) and subject to linkage drag.
Enhanced by genomics and marker-assisted selection.
Effective, but slow, and limited range of accessible genes/traits.
Generally unregulated (exception: Canada’s “novelty” criterion).

4. Plant Genetic Modification Technologies
Induced mutations – involves introducing mutations into the plant genome, generally randomly, via chemical mutagens, transposons or irradiation.
Over 2000 cultivars have induced mutagenesis step in pedigree.
Undirected and many unknown changes also introduced.
“Needle in haystack” issue if searching for a specific mutation.
TILLING populations have made it more efficient if target gene is known.
Generally unregulated (exception: Canada’s “novelty” criterion).

5. Plant Genetic Modification Technologies
Transformation – involves transfer of genes directly into recipient cells without sexual crossing.
Introduces genes directly as recombinant DNA.
Genes can be from any source, including synthetic, and endogenous genes can be silenced (RNAi).
“Transgenesis” – from other species; “cisgenesis” or “intragenesis” – from same species; cisgenics may or may not be regulated in US depending on methods used.
Can be Agrobacterium-mediated or by direct introduction (biolistics, etc.).
Insertion sites essentially random and expression levels can vary.
Regulated if Agro is used (in US). In EU, product remains regulated even if transgene is no longer present (i.e., null segregants still regulated).

6. Plant Genetic Modification Technologies
Doubled haploids – modifications to chromosome recombination or assortment
“Reverse breeding” – blocks recombination, enabling selection of whole chromosomes from the parents; enables novel creation of hybrids, whole chromosome breeding, etc. Inducing transgene can be eliminated by segregation.
Centromere-mediated chromosome elimination (CCE) – enable elimination of inducer chromosomes and creation of doubled haploids; can be engineered or natural.
Unregulated per se, but regulated if transgenes are used or are present.

7. Plant Genetic Modification Technologies
Genome editing or precision breeding – involves introducing site-specific mutations, deletions or insertions into the genome. Depending on the type of technology used, mutations can either be restricted to one or a few nucleotides or involve the insertion of larger pieces of DNA.
Sequence-specific nucleases: meganucleases, zinc finger nucleases, TALENs and CRISPR/Cas9.
Takes advantage of endogenous DNA repair mechanisms.
Single bases up to entire genes can be changed.
Can reproduce mutations with no foreign DNA left in the recipient plant.
Changes are targeted to a specific DNA location (with implications for event-by-event regulation).
Much higher efficiencies (e.g., with CRISPR, potentially up to 40% and in both alleles simultaneously).
Regulatory status under discussion; no in US for deletions, case by case for others; probably yes in EU.

8. Three Agencies Regulate Biotech Crops in the US
The US Dept. of Agriculture determines whether the crop is safe to grow based on authority to regulate plant pests. For example, is it a threat to become a weed; what are its growth and flowering characteristics? Were any plant pests used in its development (e.g., Agrobacterium)?
The Food and Drug Administration determines whether the crop is safe to eat. Is it substantially equivalent to other crops with respect to composition, nutrition, allergenicity, digestibility, etc.?
The Environmental Protection Agency regulates crops that have pesticidal properties. Are they safe for humans, for non-target organisms, and for the environment?

9. Letters of Inquiry on Regulated Status
USDA does not regulate GE unless the product itself is a potential plant pest or is used in the process (CFR 340).
In the US, the USDA, FDA and EPA are authorized to regulate certain transgenic products. However, products that do not meet the legal definition of a “regulated article” are eligible to bypass regulatory oversight of the USDA.
A GE organism’s regulatory status may be determined by sending a signed ‘Letter of Inquiry’ to receive an evaluation by the USDA’s Biotechnology Regulatory Service (BRS).
State of biotechnological research has therefore surpassed capacity with which current regulatory framework can effectively work

10. Letters of Inquiry vs FONSI Determinations
State of biotechnological research has therefore surpassed capacity with which current regulatory framework can effectively work
Camacho et al. (2014) Genetically engineered crops that fly under the US regulatory radar. Nat Biotech 32:

11. Letters of Inquiry on Regulated Status
State of biotechnological research has therefore surpassed capacity with which current regulatory framework can effectively work
GE crops modified by targeted deletions, during which no ‘plant pest’ genetic information is incorporated into the host genome, were determined to fall outside of the scope of 37 CFR Part 340; GE crops modified by targeted insertions would have to be reviewed on a case-by-case basis to determine regulatory status.
Camacho et al. (2014) Genetically engineered crops that fly under the US regulatory radar. Nat Biotech 32:

12. Technologies and Regulation
Item
USA
Canada
Europe
New Zealand
Transgenic
Yes
Cisgenic
Mutant lines No
Transgenic in pedigree but not in plant
Transformed without Agrobacterium
Genome editing: deletions ?
Genome editing: mutations or insertions
Case by case
Likely

13. All Technologies and Regulatory Status
Alex Camacho, PIPRA, UC Davis, January 2015, unpublished data