Genetic Engineering in Agriculture: Science, Policy & Law UC Davis, June 13, 2017

Kent J. Bradford

Department of Plant Sciences Seed Biotechnology Center

University of California, Davis kjbradford@ucdavis.edu

Plant Genetic Improvement: Techniques and Regulatory Status

Domestication: The Original Crop Biotechnology

Humans have made dramatic changes in plants during the process of domestication that have resulted in the crops that we rely on today.

The majority of these changes had already occurred before scientific breeding began.

UCDAVIS

Early corn Modern corn Teosinte and early progenitors of corn

Photo courtesy of Monsanto

Plant Breeding Is Genetic Modification

Through the application of genetics, plant breeders have directed changes in the genetic characteristics of crops to achieve a desired result.

UCDAVIS

• Grafting – involves utilizing traits from two varieties/species by grafting a scion variety on to a rootstock variety.

Grafting

• Sexual crosses – involves transfer of genes via sexual crossing and subsequent chromosomal segregation and recombination.

Goldberg et al. (1994) Science 266: 605-614.

Egg

Sexual Crosses

• Induced mutations – involves introducing mutations into the plant genome, generally randomly, via chemical mutagens, transposons or irradiation.

Mutations

• Over 3200 crop varieties, including • CalRose semi-dwarf rice with higher yields • Heat tolerance and early maturity in cotton • Multiple disease resistances in tomato • Gold Nijisseiki disease-resistant Japanese pear • High-yielding and short barley for brewing industry • Seedless watermelon • Peanuts with tougher hulls • Canola with healthy fatty acid composition • Ruby Red grapefruit

• Transformation – involves transfer of genes directly into recipient cells without sexual crossing using recombinant DNA methods.

Genes “grafted” into the DNA of the host plant.

Transformation – Gene Grafting

Agrobacterium

• Gene silencing (RNA interference) – suppress or silence expression of a specific gene by expressing an antisense or a hairpin double-stranded version of the transcript sequence.

Gene Silencing

+ siRNA

MicroRNA

Hairpins

Transcription

Antisense RNA

FlavrSavr 1994

Virus-resistant Crops By 1995, Papaya ringspot virus had devastated the Hawaiian papaya industry.

Photos by D. Gonsalves

Virus resistance introduced via rDNA techniques has re-established the industry in Hawaii.

New Approvals in the US

Arctic Apple (Okanagan) • Non-browning after cutting • Used RNAi to silence a polyphenol

oxidase

Innate Potato (Simplot) • Reduced bruising (30% waste) • Reduced browning upon frying • Reduced acrylamide production

• Example: Host-induced gene silencing (HIGS)

Disease Resistance

Proof of concept: Bremia lactucae, lettuce downy mildew (related to Phytophthora spp.) Govindarajulu et al. (2014). Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce. Pl. Biotech. J. DOI: 10.1111/pbi.1230.

Primary targets: Puccinia striiformis, wheat stripe rust and P. graminis, wheat stem rust (UG99)

PIs: Richard Michelmore, Jorge Dubcovsky Researchers: Manju Govindarajulu, Dario Cantu & Jin-Ying Gou Funding: Bill & Melinda Gates Foundation

Plant Genetic Modification Technologies

* Incompatible Interaction

Susceptible Resistant Susceptible Resistant

RNAi HAM34 RNAi GUS (control) WT (control) Diana* (control)

M. Govindarajulu et al. (2014) Plant Biotech. J. doi: 10.1111/pbi.12307

Transgenic T3 lettuce seedlings expressing RNAi HAM34 are resistant to B. lactucae (8 dpi)

HIGS offers a novel control strategy for B. lactucae in lettuce and potentially other pathogens of additional crops such as stem rust of wheat, citrus greening, etc.

• Genome editing – based on cutting DNA and allowing natural repair of the cut ends.

Genome Editing

DNA sequences • DNA sequence to be edited

• Cut DNA at site to be edited

ATGGGATCC

• Errors or deletions on repair • Replace one allele with another • Insert genes into specific sites

Provide a copy of DNA needed to replace gap

Desired change at specific site ATGGCATCC

ATGGGATCC

ATGGG…ATG...ATCC

• 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 CRISPR/Cas9

Genome Editing – Precision Breeding

• Zinc finger nucleases (ZFN) and Transcription Activator-line Effector Nucleases (TALENs) – Proteins recognize specific DNA sequences to target nucleases.

Targeted Nucleases

Moore et. al, 2012. PLoS ONE. 2012;7:e37877

Specific proteins that can recognize the target site must be designed and synthesized.

• CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 – an RNA guides the nuclease (Cas9) to cut DNA at a specific location.

CRISPR-Cas9

E. Pennisi. 2013. Science 341: 833-836

Nuclease

• Gene editing methods: • Allow site-specific modifications. • Can change specific bases to modify alleles, e.g., replicate mutations or

wild alleles in cultivars. • Can modify all alleles in a genome at the same time (e.g., for polyploids). • Can modify multiple different genes at the same time by expressing

multiple guides and templates. • Higher efficiency compared to transformation. • Leaves no “foreign” DNA behind. • Short base modifications or deletions would be indistinguishable from

mutations. • Can transfer larger segments (genes) as well as make base

modifications. • Regulatory situation case by case, but some cases not regulated.

Gene Editing Methods

Maize

Rice

Tomato

Seedless grapes, mandarins, tangelos, watermelons, …….

Tomato

While selecting for color in tomato, flavor-related genes have been left behind. Now that specific genes for better flavor have been identified, they could be specifically modified to match the genes that are present in more flavorful varieties without losing valuable production and quality traits.

modern

modern

color

flavor

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?

Regulatory Costs

Kalaitzandonakes et al. (2007) Nature Biotech. 25: 509-511.

Bacterial Leaf Spot Disease

• Xanthomonas perforans, aka Xanthomonas campestris pv vesicatoria

• 92% of FL acreage and 40% of U.S. tomato acreage affected

• Effective genetic resistance not available

• Chemical control (copper) inadequate

Two Blades Foundation

Transfer of Disease Resistance In collaboration with Two-Blades Foundation, UC Berkeley and the University of Florida, bacterial spot (Xanthomonas) resistance was transferred from pepper to commercial fresh market tomatoes.

Horvath et al. (2012). Transgenic resistance confers effective field level control of bacterial spot disease in tomato. PLoS ONE 7, e42036.

Bs2

Replace ineffective copper sprays

VF36

VF36-Bs2

Destruction of Golden Rice Trials at IRRI

On April 27, 2013, protesters bussed in by anti-GMO groups attacked and destroyed research plots of Golden Rice at the International Rice Research Institute in the Philippines.

http://www.slate.com/blogs/future_tense/2013/08/26/golden_rice_attack_in_philippines_anti_gmo_activists_lie_about_protest_and.html

Orange Petunias

Transgenic plants were made in 1987 (or 1995) but not officially released or subjected to regulatory approval. Many current varieties were recently found to have this transgene in them, unbeknownst to the companies breeding and selling them. Regulatory agencies may require them all to be destroyed.

Regulated Status and Modification Methods

• 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).

Mushrooms

Nature 532: 293 (21 April 2016)

Letters of Inquiry vs FONSI Determinations

Camacho et al. (2014) Genetically engineered crops that fly under the US regulatory radar. Nat Biotech 32: 1087-1091.

How Will Gene Editing Be Regulated?

www.nbtplatform.org

Federal comment period for FDA and Part 340 (plant and animal) due June 19, 2017.

Technologies and Regulation

Item USA Canada Europe New

Zealand Transgenic Yes Yes Yes Yes

Cisgenic Yes Yes Yes Yes

Mutants No Yes No No

Transgenic in pedigree but not in plant No No Yes No

Transformed without Agrobacterium No Yes Yes Yes

Genome editing: deletions No ? ? No

Genome editing: mutations or insertions Case by case

Likely Yes ?

Regulatory Status of All Technologies

Alex Camacho, PIPRA, UC Davis, January 2015, unpublished data

Gene Delivery Systems

Bacterially-Mediated

Agrobacterium-Mediated

Transbacter™ Suite Ensifer-Mediated

Biolistics

Transgrafting

Null Segregants (NS)

Reverse Breeding (RB)

Centromere-Mediated

Chromosome Elimination (CCE)

Cis/Intragenesis

Genome Editing

RNA-dependent DNA Methylation (RdDM)

Engineered Nucleases

Zinc –Finger Nuclease (ZFN)

Meganuclease (I-CreI)

Transcription Activator-Like

Effector Nucleases (TALENs)

CRISPR-Cas9 Systems

RNAi

TILLING

Regulated Non-Regulated*

Case-by-Case Review

Not Yet Determined

Color Key:

*Not regulated by USDA; assumes no $340.2 Plant Pests are used as genetic sources or vector agents in transformation.

Labeling of GM Foods

• Previously, as all GM crops had been thoroughly reviewed and determined to be safe, no labeling was required on foods made from them.

• In 2016, a federal GM food labeling law was passed.

• Requires a label on foods produced using transgenic methods.

• Preempts state regulations. • Two-year period to develop and enact specific

regulations as to how the law will be implemented.

Misleading Labeling of non-GM Foods

Summary

• Virtually all of our foods have been genetically modified from their original form.

• Modern genetic improvement methods are extensions of breeding that make it more precise and targeted.

• After 20+ years of safe use, do we still need such a precautionary approach to regulation of these methods?

• Implementation of the GMO labeling law will lock in the process-based regulatory approach for the forseeable future.

Kent J. Bradford Department of Plant Sciences Seed Biotechnology Center

University of California, Davis kjbradford@ucdavis.edu

Thank you!