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A case study: Cisgenic barley and wheat for animal feed Preben Bach Holm Aarhus 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 5 G-2, June 21, 2011

Project Cisgenic barley and wheat for animal feed Aim: 1) To develop new generations Project Cisgenic barley and wheat for animal feed Aim: 1) To develop new generations of genetically modified feed barley and wheat based on the concept of cisgenesis 2) To assess if such crops provide environmental benefits, have economic advantages and are perceived as useful and ethical acceptable by the Danish citizens. 3) 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.

Cisgenesis criteria 1) Only utilize genes for genetic modification (genomic clones) from the same Cisgenesis criteria 1) Only utilize genes for genetic modification (genomic clones) from the same species and species with which it can intercross 2) 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

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

Phytic acid and phytases O Zn O ++ H Ca O O P O Phytic acid and phytases O Zn O ++ H Ca O O P O O 5 ++Mg H P O O 4 3 O H 6 O O P O O 1 ++ Ca P O O O HO P O O Fe O H O P O ++ H 2 H ++ • 70 -80% of the phosphate reserves in seeds are bound as phytic acid. • Phytic acid can only be degraded by specific enzymes – 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

Phosphate: An important nutrient and limited resource • Monogastric animals like pigs, chicken and Phosphate: An important nutrient and limited resource • 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

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

Frequency of cisgenic lines Frequence of cisgenic lines= Frequency of GM lines * Frequency 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

Public perception and ethical issues The main results of the analyses were that cisgenic Public perception and ethical issues 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

Down-stream handling and segregation On the farm Storage facility on farm Non cisgenic field Down-stream handling and segregation On the farm Storage facility on farm Non cisgenic field Animal production Cisgenic field Grain elevator Slurry / manure Field on neighbour farm Outside the farm Haastrup, Nielsen, Hauge Madsen and Gylling 2011, in preparation

Consequences of different levels of regulation for fodder wheat and barley 1. Cisgenic grain Consequences of different levels of regulation for fodder wheat and barley 1. Cisgenic grain is handled as a Gmo (full regulation) 2. Cigenic grain has a lower level of risk assessment and segregation requirements (deregulated) 3. Cisgenic grain is handled as a non-GM fodder grain variety (No regulation) 1. 2. 3. 4. 5. 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

Break even analysis Estimated potential price reduction on premix per 1 ton of feed Break even analysis Estimated potential price reduction on premix per 1 ton of feed mix: Scenario 1: 7, 80 kr. /ton Scenario 2: 20, 10 kr. /ton Scenario 3: 32, 30 kr. /ton (- phytase) (- phytase – 50% MCP) (- phytase - MCP) 1) Substitution of phytase in on farm feed mix is economic competitive (after coexistence costs) if cisgenic wheat is used as the only grain component. 1) If all MCP or part of it can be substituted in the on farm feed mix by cisgenic grain with high phytase activity both cisgenic barley and wheat will be economic competitive (after coexistence costs) Gylling et al. 2011. In preparation

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

Gene pool concept in crop breeding Primary gene pool (GP-1): Varieties of the same Gene pool concept in crop breeding Primary gene pool (GP-1): Varieties of the same species that can intermate freely 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 JMT de Wet. 1971. Toward a Rational Classification of Cultivated Plants. Taxon 20: 509 -517. and Wikipedia

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Cisgenic barley and wheat for animal feed A project funded by the Danish Directorate 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)

Transformation in Wheat Promoter Genomic Ferritin clone 1 DX 5 Glutenin 1 kb. Nos Transformation in Wheat Promoter Genomic Ferritin clone 1 DX 5 Glutenin 1 kb. Nos Exon Intron A We have introduced extra copies of the genomic sequence of the most active homoeoallele of the Ta. Fer 1 gene into wheat by transformation using the high molecular weight glutenine 1 Dx 5 promoter for driving endosperm specific expression. B Biolistic transformed ferritin transgenic plants, (A) on selection media and (B) in pots.