-

1. Crop plants with improved culture and
quality traits for food, feed and other uses
Peter ROGOWSKY
Plant Reproduction and Development, Lyon,
France

2. (1) Crop plants with improved culture
and quality traits
The project

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. (2) Crop plants with improved
nutritious components
Examples from the literature

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. 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. • 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. (3) Conclusion

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. 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. 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. 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. Thank you
for your attention