39
New Plant Breeding Techniques March 2015, Singapore
New Plant Breeding Techniques
March 2015, Singapore
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)
IGTC Business Plan 2015-2018
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 varietiesSource: 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
• 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)
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 developmentCisgenics/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 difficultTechniques Involves a GM technique? Produces an intermediate
product that is a GMO?Offspring are GMOs?
Cisgenesis/intragenesis Yes*(B) / Yes -- Yes *(B) / Yes
Reverse Breeding Yes Yes No * (A)
Agroinfiltration Yes Questionable No
Grating (non GM-scion /GM-rootstock)
No Yes No
RNA-dependent DNA methylation
No – nucleic acid molecules not inserted into genomeYes – nucleic acid molecules inserted into genome
No
Yes
No*(B)
No
Oligo-directed mutagenesis Yes No No
Zinc-finger nucleases (mutagenesis
Yes No No*(C)
* Uncertainty about legal interpretation of the definitionA: Offspring of GMO’s =>GMO’s? / B: Unclarity on ‘altering’ of genetic material / C: Recombinant nucl. acid molec. if not inserted into genome…
Source: Advisory Committee of Releases in the Environment (ACRE)
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 case *
Likely Yes ?
Source: van Deynze, 2014
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
NPBTs - Concerns
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
techniquesCrucial 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 - BIACBusiness and Industry Advisory Committee: Innovative improvements of existing methodsCharacteristics of plant that determine safetyPublic, private & scientist alike have sign opport. to employ NPBTs
in their breeding programsAdoption of techniques will depend on regul. requirementsAll gov’s encouraged to adopt globally harmonized approach &
avoid unnecessary oversightGov’s encouraged to provide predictable, timely guidance on
oversight of NPBTs => foresee appropriate investment & commercialization
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
NPBTs - Conclusion
Thank you for your attention
Back Up slides
NPBT’s : Four classes of techniques
1: GM is used as a tool to facilitate breeding2: Plants obtained by combining GM and non-GM plant by grafting => chimeric plants3: Tool to introduce new, but in germplasm occurring characteristics => cis- & intra-genesis4: 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 GMNo 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 speciesCisgenesis: 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 patternOligonucleotide-mediated mutation inductionSo far only described for amino-acid substitutions into ALS-gene =>herbicide resistanceEnd result similar to plant obtained through mutation breedingNo 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
