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Crop plants with improved culture and
quality traits for food, feed and other uses
Peter ROGOWSKY
Plant Reproduction and D...
(1) Crop plants with improved culture
and quality traits
The project
Consortium
• Acronyme
– Genome engineering improvement for useful plants of a sustainable agriculture
• Funding
– 6 M€ (Fr...
Plant species
• 9 cultivated plant species
– 4 crop
– 2 vegetable
– 1 fruit
– 1 forestry
– 1 ornamental
• 3 model species
...
Improvement of cellular engineering
• Increased transformation rates
– Apple
– Poplar (novel in planta technique)
– Rose (...
New ways to introduce editing tools into the cell
• Establishment/reactivation of
protoplast transformation
– Interest
• R...
3 ways to edit genomes
• Successful establishment
– Targeted mutagenesis (12/12)
– Genome editing (2/12)
– Base editing (2...
Marker genes
Paris, June 28th, 2018 Genome Editing Conference 8
 Visual marker systems are helpful to establish and optim...
Breeding tools
Paris, June 28th, 2018 Genome Editing Conference 9
 Modifications of plant reproduction present an interes...
Quality traits
Paris, June 28th, 2018 Genome Editing Conference 10
 Metabolic pathways provide numerous targets for genom...
Flowering time
Paris, June 28th, 2018 Genome Editing Conference 11
 Modification of flowering time can help mitigate clim...
Abiotic stress tolerance
Paris, June 28th, 2018 Genome Editing Conference 12
 Knockin without selective marker requires o...
Disease resistance
Paris, June 28th, 2018 Genome Editing Conference 13
 Editing rather than inactivation of host factors ...
Summary
• Key numbers
– 22 TALEN couples introduced into plant cells
– 185 Guide RNAs introduced into plant cells
– 316 Mo...
(2) Crop plants with improved
nutritious components
Examples from the literature
Low gluten wheat
Paris, June 28th, 2018 Genome Editing Conference 16
 Genome editing can provide plant products with impr...
Lycopene enriched tomato
Paris, June 28th, 2018 Genome Editing Conference 17
 Fruits are an important part of human diet ...
• Results (soybean)
– Double knockout necessary for effect
– 4-fold increase in oleic acid, strong decrease in linoleic
ac...
(3) Conclusion
Traits accessible by genome editing
Paris, June 28th, 2018 Genome Editing Conference 20
• All traits partially or entirely...
Conclusion (i)
• Promises/claims
– Genome editing is necessary to feed a
growing world population
– Genome editing will re...
Conclusion (ii)
• Genome editing has become a major tool in basic research
• Genome editing will have a major impact on ag...
Acknowledgements
Peter Rogowsky, Philippe Vergne INRA Lyon
Anne-Marie Chèvre, Jean-Eric Chauvin INRA Rennes
Fabien Nogué, ...
Thank you
for your attention
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Crop plants with improved culture and quality traits for food, feed and other uses

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Crop plants with improved culture and quality traits for food, feed and other uses

  1. 1. Crop plants with improved culture and quality traits for food, feed and other uses Peter ROGOWSKY Plant Reproduction and Development, Lyon, France
  2. 2. (1) Crop plants with improved culture and quality traits The project
  3. 3. Consortium • Acronyme – Genome engineering improvement for useful plants of a sustainable agriculture • Funding – 6 M€ (French National Research Agency) – 7 years (2012 to 2019) • Partnership – 10 public and 4 private labs – 11 life science and 3 social science labs • Objectives – Implement genome modification techniques in crops – Improve the efficiency and throughput of crop transformation – Provide proof of concept for improved traits useful for agriculture – Acquire and maintain technical know-how in France – Assess the economic, ethical and legal impact Paris, June 28th, 2018 Genome Editing Conference 3  Long term funding for research in confined environments
  4. 4. Plant species • 9 cultivated plant species – 4 crop – 2 vegetable – 1 fruit – 1 forestry – 1 ornamental • 3 model species Paris, June 28th, 2018 Genome Editing Conference 4  Genome editing is applicable to a wide range of cultivated plant species Species Category Wheat Field crop Rice Field crop Maize Field crop Rapeseed Field crop Tomato Vegetable Potato Vegetable Apple Fruit Poplar Forestry Rose Ornamental Physcomitrella Model Brachypodium Model Arabidopsis Model
  5. 5. Improvement of cellular engineering • Increased transformation rates – Apple – Poplar (novel in planta technique) – Rose (from 3% to 30%) • Extension to elite lines – Success in 5 species – Rootstock for apple Paris, June 28th, 2018 Genome Editing Conference 5  Cellular engineering remains very species and genotype dependent  Cellular engineering is a bottleneck of genome editing Species Category Introduction into lab genotypes Introduction into elite genotypes Wheat Field crop Low ND Rice Field crop High ND Maize Field crop Low Yes Rapeseed Field crop Medium ND Tomato Vegetable High ND Potato Vegetable High Yes Apple Fruit Low (improved) Yes Poplar Forestry Low (improved) Yes Rose Ornamental Medium (improved) Yes Physcomitrella Model High ND Brachypodium Model Low ND Arabidopsis Model Medium ND
  6. 6. New ways to introduce editing tools into the cell • Establishment/reactivation of protoplast transformation – Interest • Rapid test of CRISPR/Cas design • Rapid test of novel tools • Transient Cas9 expression • DNA-free genome editing – Drawbacks • Regeneration • Biolistics Paris, June 28th, 2018 Genome Editing Conference 6  A regain of interest in alternatives to Agrobacterium-mediated transformation Species Category Agro- bacterium Proto- plasts Biolistics Wheat Field crop Yes ND Yes Rice Field crop Yes Yes ND Maize Field crop Yes Starting ND Rapeseed Field crop Yes ND ND Tomato Vegetable Yes Yes Yes Potato Vegetable Yes Yes ND Apple Fruit Yes ND ND Poplar Forestry Yes Starting ND Rose Ornamental Yes ND ND Physcomitrella Model ND Yes ND Brachypodium Model Yes ND ND Arabidopsis Model Yes Yes ND
  7. 7. 3 ways to edit genomes • Successful establishment – Targeted mutagenesis (12/12) – Genome editing (2/12) – Base editing (2/12) Paris, June 28th, 2018 Genome Editing Conference 7  True genome editing (SDN2) remains very challenging in plants Species Category Targeted mutagenesis Genome editing Base editing Wheat Field crop Yes ND ND Rice Field crop Yes In progress ND Maize Field crop Yes ND In progress Rapeseed Field crop Yes ND ND Tomato Vegetable Yes Yes Yes Potato Vegetable Yes In progress In progress Apple Fruit Yes ND ND Poplar Forestry Yes In progress ND Rose Ornamental Yes ND ND Physcomitrella Model Yes Yes Yes Brachypodium Model Yes In progress ND Arabidopsis Model Yes ND ND  Base editing provides a more limited but efficient alternative
  8. 8. Marker genes Paris, June 28th, 2018 Genome Editing Conference 8  Visual marker systems are helpful to establish and optimize genome editing Species Apple, rice, wheat, poplar, tomato, Physcomitrella Gene targeted PDS (Phytoene desaturase) CAO (Chlorophyllide-a oxygenase) GUS (β-glucuronidase) DFR (Dihydroflavonol 4-reductase) APT (Adenine phosphoribosyltransferase) Modification Targeted mutagenesis (SDN1), genome editing (SDN2) Trait Bleached leaves Yellow leaves Loss of blue histochemical staining Loss of purple color Survival on 2-Fluoroadenine Use Establish and optimize genome editing Comment APT is a selective marker Status Finished Laboratories INRA and CIRAD (E. Chevreau, E. Guiderdoni, P. Barret, A. Déjardin, M. Mazier, F. Nogué) Apple Rice, wheat Poplar Tomato Physcomitrella
  9. 9. Breeding tools Paris, June 28th, 2018 Genome Editing Conference 9  Modifications of plant reproduction present an interest for plant breeding Species Maize Gene targeted ZmNLD (Not like dad) = ZmPLA1 = ZmMTL Modification Targeted mutagenesis (SDN1) Trait Haploid plants Use Rapid production of pure homozygous lines Asexual propagation of crops Comment Two of 4 mutations to replace meiosis by mitosis; similar publications by 2 other labs Status Finished Laboratory INRA-Lyon (P. Rogowsky) Limagrain (J.-P. Martinant Gynogenesis => Development of haploid plants containing only maternal genetic material • Results – Novel mutants inducing gynogenesis upon pollination of standard maize lines WTWT WT nld F1 F1
  10. 10. Quality traits Paris, June 28th, 2018 Genome Editing Conference 10  Metabolic pathways provide numerous targets for genome editing Species Potato Gene targeted StGBSS (Granule bound starch synthase) Modification Targeted mutagenesis (SDN1) Trait Starch composed only of amylopectin Use Recovery after sport, additive in food and glue industry Comment Similar work published in other potato varieties and in maize Status Potato tubers with reduced amylose levels Laboratory INRA-Ploudaniel and Germicopa (J.-E. Chauvin, P. Devaux) Starch = Amylopectin + Amylose (branched) (linear) from Zimmermann and Snow (2012) An introduction to nutrition WT
  11. 11. Flowering time Paris, June 28th, 2018 Genome Editing Conference 11  Modification of flowering time can help mitigate climate change and enhance crop rotation Species Apple Gene targeted MdTFL1 (Terminal flower) Modification Targeted mutagenesis (SDN1) Trait Rapid flowering Use Shortening of the life cycle in perennial species, some of which flower only after several years Comment Flowers have some aberrations Terminal flower hampers vegetative development Status Finished Laboratory INRA-Angers (E. Chevreau) Flower bud Flower Flower parts WT GE  Genome editing is a helpful tool for fast breeding
  12. 12. Abiotic stress tolerance Paris, June 28th, 2018 Genome Editing Conference 12  Knockin without selective marker requires optimization of all steps Species Rice Gene targeted OsSAP (Stress associated protein) Modification Genome editing (SDN2) Trait Tolerance to salinity Use Rice culture in lower basins or on marginal land Comment Status Validation of efficient cleavage, knockin to come Laboratory CIRAD-Montpellier (E. Guiderdoni) from Zhao et al. (2016) FPLS 5:764 • Expected phenotype Salt stress No stress
  13. 13. Disease resistance Paris, June 28th, 2018 Genome Editing Conference 13  Editing rather than inactivation of host factors may help to maintain agronomic performance Species Tomato Gene targeted eIF4E (Elongation initiation factor 4E) Modification Genome editing (SDN2) Trait Resistance to potyviruses Use Sustainable plant protection Comment Published examples in other species concern loss-of-function, not genome editing Status Achieved via intermediary step with selective marker (subsequent excision) Laboratory INRA-Avignon (M. Mazier) Intron 1Exon 1 P Marker T Intron 1Exon 1 P Marker T Intron 1Exon 1 Repair matrix Genome (sensitive) Genome (resistant) from Mazier et al. (2011) PlosONE 6e29595 eIF4E expression
  14. 14. Summary • Key numbers – 22 TALEN couples introduced into plant cells – 185 Guide RNAs introduced into plant cells – 316 Molecular constructs introduced into plant cells – 7505 Plants regenerated Paris, June 28th, 2018 Genome Editing Conference 14  Genome editing has become an indispensable tool in basic research  Genome editing has been used successfully in a wide range of plant species for diverse traits  Proof of concept has been provided for traits of agronomic interest https://www6.inra.fr/genius-project/
  15. 15. (2) Crop plants with improved nutritious components Examples from the literature
  16. 16. Low gluten wheat Paris, June 28th, 2018 Genome Editing Conference 16  Genome editing can provide plant products with improved health characteristics Species Wheat (2 bread wheat and 1 durum wheat) Gene targeted 45 TaGli-2 genes (α-gliadin); conserved region adjacent to allergenic 33-mer domain Modification Targeted mutagenesis (SDN1) Trait Low gluten content Use Low allergen food, avoid development of coeliac disease and non-coeliac gluten sensitivity (7% Western population) Comment Loss of α-gliadin genes influences bread making quality Laboratory Francisco Barro, IAS-CSIC Cordoba, Spain • Results – Mutations in up to 35/45 α-gliadin genes – 32%–82% reduction in α-gliadin, – Indirect effect on γ-gliadins (25%–94% reduction), no but not ω-gliadins – No off-target (expected effects) – Up to 85% reduction in immunoreactivity (R5 = all gluten = food industry, G12 = 33-mer domain) – Variability in SDS sedimentation (prediction of bread making quality)
  17. 17. Lycopene enriched tomato Paris, June 28th, 2018 Genome Editing Conference 17  Fruits are an important part of human diet and a target for biofortification Species Tomato Gene targeted SlSGR1 (Stay green 1), an inhibitor of PSY1 (x 2) SlLCY-B1, SlLCY-B2 (Lycopene β-cyclase) SlLCY-E (Lycopene α-cyclase) SlBlc (Lycopene β-cyclase and ε-cyclase) Modification Multiplex targeted mutagenesis (SDN1) Trait Enriched lycopene content Use Lycopene is an antioxidant that may prevent the onset of certain cancers Comment Possible effects on whole plant performance need to be assessed Laboratory Hongliang Zhu, College of Food Science & Nutritional Engineering, CAU, Beijing, China • Results – Various efficiencies for the 6 target sites – 5 classes (24 single to quadruple mutants) – Up to 5.1-fold increase in lycopene content – Concomitant increase in β-carotene content – No off-target (expected effects, first 2 candidates)
  18. 18. • Results (soybean) – Double knockout necessary for effect – 4-fold increase in oleic acid, strong decrease in linoleic acid (12-fold) and linolenic acid – Stronger effects with triple mutant including FAD3A (follow up by Demorest et al., 2016) • Results (camelina) – Range from 1 to 6 mutations – Triple mutants strongly affected in growth – Increase in oleic acid (C18:1), decrease in linoleic acid (C18:2) High oleic acid soybean and camelina Paris, June 28th, 2018 Genome Editing Conference 18  Modification of seed oil composition is a lever for improved food and feed Species Soybean and camelina Gene targeted GmFAD2-1A and GmFAD2-1B genes (Fatty acid desaturase 2) CsFAD2-1, CsFAD2-2 and CsFAD2-3 genes Modification Targeted mutagenesis (SDN1) Trait Change in oil composition Use Longer shelf life without artificial hydrogenation of soybean oil Health benefit of monounsaturated fatty acids Comment Other crops (olive) naturally contain high levels of oleic acid; ω-9 fatty acids not essential Laboratory Feng Zhang, CALYXT, New Brighton, MN, USA Jean-Denis Faure, APT, Versailles, France
  19. 19. (3) Conclusion
  20. 20. Traits accessible by genome editing Paris, June 28th, 2018 Genome Editing Conference 20 • All traits partially or entirely determined by genetics • Upfront knowledge on the trait – Which gene(s) to modify – Which modification(s) • Complex traits more difficult than monogenic traits – Monogenic: more frequent for disease resistance, herbicide tolerance, quality – Complex: stress tolerance, nutrient use efficiency • No technical link between technique and trait  Genome editing can be used for a large panel of agronomic and quality traits conventional genetics genomic selection mutagenesis genome editing GMO crops aptitude for association abiotic stress tolerance disease resistance biomass conversion biofortification efficient use of nutrients agroecology climate change less pesticides bioenergy health less intrants
  21. 21. Conclusion (i) • Promises/claims – Genome editing is necessary to feed a growing world population – Genome editing will revolutionize plant breeding – Hundreds of genome edited crops will be commercialized in the years to come – Genome editing is nowadays routine in plant science Paris, June 28th, 2018 Genome Editing Conference 21 • Reality – Genome editing is only one of many tools to achieve food security – Genome editing greatly facilitates genetic improvements that would have been more cumbersome with existing tools – In countries opting for deregulation, improvements based on existing knowledge are likely to see the market – Targeted mutation (and base editing) but not genome editing is routine in a few genotypes of many species Headlines are often disconnected from the reality in labs
  22. 22. Conclusion (ii) • Genome editing has become a major tool in basic research • Genome editing will have a major impact on agriculture under 5 conditions – Sufficient knowledge on genes <=> traits – Sharply increased efficiency of true genome editing (SDN2) – Accessibility of breeding material (elite genotypes) to genome editing – Access to genome editing technologies for SME (licensing at reasonable cost) – Deregulation Paris, June 28th, 2018 Genome Editing Conference 22  A long way from gene inactivation in labs to edited crop plants on the market
  23. 23. Acknowledgements Peter Rogowsky, Philippe Vergne INRA Lyon Anne-Marie Chèvre, Jean-Eric Chauvin INRA Rennes Fabien Nogué, Pierre Hilson INRA Versailles Annabelle Déjardin INRA Orléans Elisabeth Chevreau, Laurence Hibrand-Saint Oyant INRA Angers Marianne Mazier INRA Avignon Pierre Barret INRA Clermont-Ferrand Emmanuel Guiderdoni CIRAD Montpellier Christophe Sallaud Biogemma Chappes Mireille Matt INRA Grenoble Jean-Philippe Pierron University Lyon 3, Lyon Pierre Devaux Germicopa, Quimper Séverine Foucrier Delbard, Malicorne Alain Toppan Vilmorin, Toulouse
  24. 24. Thank you for your attention

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