A case study: Cisgenic barley and wheat for animal feed Preben Bach Holm Aarhus University

1. A case study: Cisgenic barley and wheat for animal feed Preben Bach Holm Aarhus University Science and Technology Dept. of Molecular Biology and Genetics Research Centre Flakkebjerg DK-4200 Slagelse Cisgenesis: What are its implications for food safety, society and economy European Parliament ASP 5G-2, June 21, 2011

2. Project • Cisgenic barley and wheat for animal feed • Aim: • To develop new generations of genetically modified feed barley and wheat based on the concept of cisgenesis • To assess if such crops provide environmental benefits, have economic advantages and are perceived as useful and ethical acceptable by the Danish citizens. • To achieve these objectives a research consortium has been assembled that integrates expertise in plant molecular biology, physiology, breeding, downstream handling and processing, economy, sociology and ethics.

3. Cisgenesiscriteria • Only utilize genes for genetic modification (genomic clones) from the same species and species with which it can intercross • The genetically modified plants should • have inserted the cisgene in the host genome with a minimum of rearrangements and outside endogenous genes • have no foreign DNA in the form of vector backbone • have no foreign DNA in the form of selection genes

4. Improvement of: • Phosphate availability • Amino acid composition • Cell wall digestibility • Starch digestibility • Mineral bioavailability Reduced N and P load on the environment

5. ++ Zn O O P O H ++ O Ca Fe O O 4 3 O P O O O O O O H O O P O 5 2 ++ P Mg HO O O O H H H 6 1 P H O ++ O P Ca O O Phyticacid and phytases • 70-80% of the phosphate reserves in seeds are bound as phytic acid. • Phytic acid can only be degraded by specific enzy-mes – phytases. • Phytases are activated at germination to ensure the supply of phosphate to the developing plantlet. • Phytic acid inhibits the uptake of zinc, calcium and iron ++

6. Phosphate: An importantnutrient and limitedresource • Monogastric animals like pigs, chicken and human cannot degrade phytic acid since they lack phytase. In consequence most of the phosphate is excreted and cause pollution • Microbial produced phytase is today added to animal feed in areas with intensive livestock production • GM cereals that produce additional phytase may be a valuable supplement to increase phytate digestibility • Many livestock farmers mix their own feed from homegrown cereals. • Organic farmers are not permitted to use microbial phytase • Phosphate is a limited non-renewable ressource that is depleted in a few decades

7. The PAPhy_agene Promoter Gene Terminator 2730 bp 2388 bp 734 bp LB Hygromycin resistance gene RB Exon Intron Co-transformation with Agrobacterium p-Soup LB x 2 PaPhy_a RB p- Clean Antibiotic resistance gene Antibiotic resistance gene

8. Frequency of cisgenic lines Frequence of cisgenic lines= Frequency of GM lines * Frequency of lines without backbone * Frequency of lines without hygromycin resistance gene * Frecuency of lines with minimal rearrangement = 10% Dosis effect of PAPhy_a

9. Public perception and ethicalissues The main results of the analyses were that cisgenic crops are acceptable for a larger part of the population than transgenic crops. Hence, 56% of the interviewed were willing to buy bread made of flour from cisgenic wheat while only 19% were willing to buy bread made from transgenic wheat. The analyses further revealed that cigenic crops are considered to be less unnatural than transgenic crops. The results are not unequivocal though since the population operated with up to five different understandings of unnaturalness and for some of these understandings cisgenis is not necessarily considered to be more natural. Perceptions of risks, benefits and unnaturalness remain, however, relevant indicators of acceptance also for cisgenic crops. This implies that the cisgenesis concept does not attend to all the concerns people have about GM crops being unnatural and risky. Miele, Lassen and Sandøe 2011. In preparation

10. On the farm Storagefacilityon farm Non cisgenicfield Down-stream handling and segregation Animal production Slurry / manure Cisgenicfield Grain elevator Field onneighbour farm Outside the farm Haastrup, Nielsen, Hauge Madsen and Gylling 2011, in preparation

11. Consequences of differentlevels of regulation for fodderwheat and barley Cisgenic grain is handled as a Gmo (full regulation) Cigenic grain has a lower level of risk assessment and segregation requirements (deregulated) Cisgenic grain is handled as a non-GM fodder grain variety (No regulation) Cleaning harvester, grain wagon, seeder and baler Cleaning grain transporter and storage containers at farm Cleaning production line at grain elevator Cleaning concrete floor Annual cleaning

12. Break evenanalysis Estimated potential pricereductiononpremixper 1 ton of feedmix: Scenario 1: 7,80 kr./ton (- phytase) Scenario 2: 20,10 kr./ton (- phytase – 50% MCP) Scenario 3: 32,30 kr./ton (- phytase - MCP) • 1) Substitution of phytase in on farm feedmix is economiccompetitive (aftercoexistencecosts) ifcisgenicwheat is used as the onlygraincomponent. • If all MCP or part of it canbesubstituted in the on farm feedmix by cisgenicgrainwithhighphytaseactivitybothcisgenicbarley and wheatwillbeeconomiccompetitive (aftercoexistencecosts) Gylling et al. 2011. In preparation

13. Wheat Triticale Trithordeum CISGENIC GROUP Rye Barley Rye x wheat x barley

14. Gene pool concept in cropbreeding Primary gene pool (GP-1): Varieties of the same species thatcanintermatefreely Secondary gene pool (GP-2):Closely related species that can intercross with GP-1 and produce at least some fertile hybrids Tertiary gene pool (GP-3):Distantly related species that can intercross with GP-1 and -2 but requires additional measures such as embryo rescue or chromosome doubling to obtain offspring. After: JR Harland and JMT de Wet. 1971. Toward a Rational Classification of Cultivated Plants. Taxon 20: 509-517. and Wikipedia

15. Thank you for your attention

16. Cisgenic barley and wheat for animal feed • A project funded by the Danish Directorate for Food, Fisheries and Agro-business (Fødevareforskningsprogrammet 2006) and Plant Biotech Denmark • Participating institutions: • COPENHAGEN UNIVERSITY • Faculty of Life Sciences, Dept of Agricultural Sciences (Jan Schjørring) • Faculty of Life Sciences, Institute of Food and Resource Economics (Peter Sandøe and Morten Gylling) • Jesper Lassen, Faculty of Life Sciences, Department of Human Nutrition (Jesper Lassen) • DANISH AGRICULTURAL ADVISORY CENTRE, THE NATIONAL CENTRE (Kathrine Hauge Madsen) • SEJET PLANT BREEDING (Kurt Hjortsholm and Lars Eriksen) • UNIVERSITY OF AARHUS, Faculty of Agricultural Sciences, Dept. of Genetics and Biotechnology (Preben Bach Holm)

17. A B 1DX5 Glutenin Nos 1 kb. Exon Intron Transformation in Wheat Promoter Genomic Ferritin clone We have introduced extra copies of the genomic sequence of the most active homoeoallele of the TaFer1 gene into wheat by transformation using the high molecular weight glutenine1Dx5 promoter for driving endosperm specific expression. Biolistic transformed ferritin transgenic plants, (A) on selection media and (B) in pots.