New Plant Breeding Techniques

March 2015, Singapore IGTC Business Plan 2015-2018 Main topics : • Grain as a hazardous product • Development of a standard for the International Movement of Grain • Global Low Level Presence Policy Initiative • Cartagena Protocol on Biosafety • New Plant Breeding Techniques (NPBTs) Different techniques

• Zinc finger nuclease (ZFN) => ZFN-1, ZFN-2 & ZFN-3 • Oligonucleotide directed mutagenesis (ODM) • Cisgenesis & intragenesis • RNA-dependent DNA methylation (RdDM) • Grafting (on GM rootstock) • Reverse breeding • Agro-infiltration (agro-infiltration “sensu-stricto”, agro-inoculation, floral dip) • Synthetic genomics Setting the scene: Literature search • 200+ publications • Grafting on GM rootstock (20+ yrs) • Other techniques: 10+ yrs • 45% EU ; 32% N. Am • EU highest: cisgenesis / intragenesis; reverse breeding; RdDM; grafting on GM rootstock • N. Am highest: ZFN-technique; ODM; agro-infiltration • Majority (81%) by public institutes • Proof of concept HT & IR traits

Source: JRC, 2011 Setting the scene: Patent search • 85+ publications – last decade • 65% US ; 26% EU • 70% private companies; 26% univ/public research inst. • EU: 83% private / 17% public • US: 68% private / 32% public • US highest: Grafting on GM rootstocks; ODM & ZFN • 50 companies: most only 1 patent => high specialisation

Source: JRC, 2011 Setting the scene: Survey plant breeding companies

• All techniques adopted by commercial breeders • Most used: ODM; cis/intragenesis; agro-infiltration • Crops at commercial development phases I-III • Less used: ZFN, RdDM; grafting on GM rootstocks & reverse breeding => mainly applied at research level • Among first commercial: HT in OSR & Corn; fungal resistance in potatoes; drought tolerance in Corn; Scab resistant apples; Potatoes with reduced amylose content

Source: JRC, 2011 NPBT’s: Drivers Fairly recent techniques, but: • NPBT’s are innovative improvements & refinements to existing breeding methods • Great technical potential => produce genetic variation • Resulting products in many cases indistinguishable /similar to existing products, produced by traditional breeding techniques • Enhance efficiency & specificity of breeding => more knowledge & understanding of final product • Adaptable to large variety of crops (incl. trees & vegetables, by researchers from all sectors (public/private; large/small)

Source: BIAC, 2014 NPBT’s: Constraints for adoption

• Efficiency (low!) => further research needed • Availability of suitable method for delivery • Regeneration • Registration costs • Low if classified as non-GMO • High if classified as GMO Cisgenesis / Intragenesis

Source: ACRE, 2014 Example Cisgenics – Apple Scab

• 85 years to conventionally breed scab resistant commercial apples • Fungus (Venturia inequalis) overcame resistance in 5 yrs • Estimated with conventional breeding: 40 yrs to breed in resistance • Cisgenic traits can reduce breeding process by 50% or more • Final product does not differ in any meaningful was from existing apple varieties

Source: Nature, 20 Aug. 2013 Directed gene modification • Oligonucleotide-directed mutagenesis (ODM): technique used to correct or to introduce specific mutations at defined sites of the genome • ODM = generic term covering several approaches and applications • Based on site-specific correction or directed mutation of target gene after introduction of chemically synthesized oligonucleotide with homology to target gene • Gene modification induced directly & exclusively via effect of oligonucleotide itself (independent of any delivery vector system)

Source: ACRE, 2014 Process of directed gene modification

Source: ACRE, 2014 Reverse breeding

• Genetic modification to facilitate production of perfectly complementing homozygous parental lines (double haploids) • Cross to generate elite heterozygous plants. • Method based on reducing genetic recombination in the elite heterozygote by inserting transgenes that suppress meiotic crossing over. • Once achieved, transgene no longer necessary Reverse Breeding

Source: ACRE, 2014 Grafting (non-GM scion onto GM rootstock)

• Non-transgenic plant tissue is grafted onto transgenic rootstock of a GM plant (e.g. genetically modified for resistance to a plant pathogen). • Non-transgenic portion of the grafted plant is thereby provided with resistance to the plant pathogen. • Endogenous mRNA enters and moves along the phloem long-distance translocation system

Source: ACRE, 2014 Grafting (non-GM scion onto GM rootstock)

Source: ACRE, 2014 DNA methylation

• Epigenetic mechanisms (such as DNA methylation) alter gene expression without changing the nucleotide sequence of the plant’s genome. • Mechanism allows plants to react to environmental stress. • Environmentally induced • RNA-dependent DNA methylation = example of epigenetic mechanism. • Methyl groups are directed to specific sequences in the genetic code by short, double-stranded RNAs (dsRNA) • Gene silencing if sequence of dsRNA molecules is identical to DNA sequence in promoter region of gene or in gene itself.

Source: ACRE, 2014 Zinc Finger Nucleases (ZFNs)

• Generating mutation in targeted (i.e. sequence-specific) manner, => more precise than random mutagenesis (conv. techniques: radiation, chem. or insertional mutagenesis (transposons, T-DNAs). • ZFNs: artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain. • Zinc finger domains can be engineered to target desired DNA sequences => enables ZFNs to target unique sequences within complex genomes. • By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.

Source: Wikipedia, 2015 Mode of action of Zinc finger nucleases 1

Source: ACRE, 2014 NPBTs – Products under development Cisgenics/Intragenics: Apple scab resistance, potato late blight resistance, drought/cold tolerant maize, fungal resistant papaya, improved forage ryegrass, a variety of vegetable crops Grafting: Citrus trees with transgenic rootstock ZFN (-1/-2/-3): Improved nutritional quality maize & canola, higher yield tomatoes, diseases resistant wheat, nematode resistance ODM: HT OSR and HT Flax NPBTs – Products under development

 Countries: AR, AU, BE, CA, IE, JP, MX, NL, CH, UK, US  Crops: Apple, Canola, Cassava, Cereal grains, Citrus, Flax, Maize, Papaya, Ryegrass, Tomato, Wheat  Developers/Users: SME’s, Academics, Industry Interpretation difficult

Techniques Involves a GM technique? Produces an intermediate Offspring are product that is a GMO? GMOs? Cisgenesis/intragenesis Yes*(B) / Yes -- Yes *(B) / Yes Reverse Breeding Yes Yes No * (A) Agroinfiltration Yes Questionable No Grating (non GM-scion /GM- No Yes No rootstock) RNA-dependent DNA No – nucleic acid molecules not inserted No No*(B) methylation into genome Yes – nucleic acid molecules inserted into Yes No genome Oligo-directed mutagenesis Yes No No Zinc-finger nucleases Yes No No*(C) (mutagenesis

Source: Advisory Committee of Releases in the Environment (ACRE)

* Uncertainty about legal interpretation of the definition A: Offspring of GMO’s =>GMO’s? / B: Unclarity on ‘altering’ of genetic material / C: Recombinant nucl. acid molec. if not inserted into genome… Differentiation in regulation

Technique To be regulated or not? Country/region=> USA Canada Europe New Zealand Transgenic Yes Yes Yes Yes Cisgenic Yes Yes Yes Yes Mutant lines No Yes No No Transgenic in pedigree but not plant No No Yes No Transformed without Agrobacterium No Yes Yes Yes Precision breeding: Deletions or small insertions No ? ? No Precision breeding: large insertions Case by Likely Yes ? case *

Source: van Deynze, 2014 NPBTs - Concerns

Risks of not accepting or regulating / asynchronously:  Authorization costs & time increase enormously  SME’s competitiveness endangered  Exodus of companies  Decrease of innovative plant breeders  Portfolio of products reduced Industry positions - ASTA American Seed Trade Association:  Governments should not differentially regulate products developed through precision breeding tools that are similar to or indistinguishable from products resulting from more traditional breeding tools.  Regulation & oversight based on sound scientific principles and proportional to the degree to which the product presents new potential safety concerns to the env. or F&F chain, and not based on the breeding process by which it was produced.  Gov’s to avoid creating trade barriers & disruptions due to non- harmonious policies Industry positions - ESA

European Seed Association:  Legal definition of GMO does not apply to most of new breeding techniques  Crucial not to hamper the application of new breeding techniques – without scientific reason – by unnecessarily subjecting them to unpredictable and excessive regulatory oversight.  Call upon EU Comm to provide legal certainty Industry positions - BIAC Business and Industry Advisory Committee:  Innovative improvements of existing methods  Characteristics of plant that determine safety  Public, private & scientist alike have sign opport. to employ NPBTs in their breeding programs  Adoption of techniques will depend on regul. requirements  All gov’s encouraged to adopt globally harmonized approach & avoid unnecessary oversight  Gov’s encouraged to provide predictable, timely guidance on oversight of NPBTs => foresee appropriate investment & commercialization NPBTs - Conclusion

 Several new techniques (20+yrs)  Great potential, but efficiency to be improved  Products similar/indistinguishable  Legal certainty needed for industry  Product that determines safety, not method  Need for globally harmonized approach Thank you for your attention Back Up slides NPBT’s : Four classes of techniques

1: GM is used as a tool to facilitate breeding 2: Plants obtained by combining GM and non-GM plant by grafting => chimeric plants 3: Tool to introduce new, but in germplasm occurring characteristics => cis- & intra-genesis 4: GM is used as a tool to make specific mutations => site-directed mutations to native genes

Source: Schaart & Visser, 2009 1: GM is used as a tool to facilitate breeding GM plants created => then used to create derivatives free of GM material • Agroinfiltration • Virus Induced Gene Silencing (VIGS) • Reverse Breeding • Accelerated Breeding Resulting plants free of DNA related to GM No additional consequences for env., food / feed

Source: Schaart & Visser, 2009 2: Plants obtained by combining GM and non-GM plant by grafting • Grafting of non-GM scion on GM-rootstock • End-product harvested on non-GM scion : free of DNA related to GM rootstock. • RNA can travel from rootstock to scion • Case by case evaluation

Source: Schaart & Visser, 2009 3: Tool to introduce new, but in germplasm occurring characteristics Genetic material originating from same of sexual compatible species  Cisgenesis: introduced DNA is unchanged natural genome fragment, containing gene of interest + its own introns & regulatory sequences (e.g. promotor and terminator sequences)  Intragenesis: allows for creation of new combination of DNA fragments. Transformation vector composed of DNA from same genome. No regulation necessary

Source: Schaart & Visser, 2009 4: GM used as tool to make specific mutations Site-directed mutations to native genes => knock-out of gene- expression or changes in gene-expression pattern  Oligonucleotide-mediated mutation induction So far only described for amino-acid substitutions into ALS-gene =>herbicide resistance End result similar to plant obtained through mutation breeding No regulation necessary

Source: Schaart & Visser, 2009

Comparison

Source: Wikipedia – Cisgenesis, 2015 Development process of GM crop

Duration & costs are industry averages. Several activities overlap. Source: Prado et. al., 2014 Safety Assessment Process

Source: Prado et. al. 2014