New Plant Breeding Techniques. March 2015, Singapore

Transcription

1 New Plant Breeding Techniques March 2015, Singapore

2 IGTC Business Plan 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)

3 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

4 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

5 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

6 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

7 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

8 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

9 Cisgenesis / Intragenesis Source: ACRE, 2014

10 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

11 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

12 Process of directed gene modification Source: ACRE, 2014

13 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

14 Reverse Breeding Source: ACRE, 2014

15 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

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

17 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

18 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 DNAbinding 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

19 Mode of action of Zinc finger nucleases 1 Source: ACRE, 2014

20 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

21 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

22 Interpretation difficult Techniques 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 /GMrootstock) RNA-dependent DNA methylation No Yes No No nucleic acid molecules not inserted into genome Yes nucleic acid molecules inserted into genome Oligo-directed mutagenesis Yes No No Zinc-finger nucleases (mutagenesis No Yes No*(B) Yes No No*(C) * 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 No Source: Advisory Committee of Releases in the Environment (ACRE)

23 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

24 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

25 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 nonharmonious policies

26 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

27 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

28 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

29 Thank you for your attention

30 Back Up slides

31 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

32 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

33 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

34 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

35 4: GM used as tool to make specific mutations Site-directed mutations to native genes => knock-out of geneexpression 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

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37 Comparison Source: Wikipedia Cisgenesis, 2015

38 Development process of GM crop Duration & costs are industry averages. Several activities overlap. Source: Prado et. al., 2014

39 Safety Assessment Process Source: Prado et. al. 2014