Development of Herbicide Tolerance Traits in Soybeans and What the Future Holds Brett Miller – Technical Product Lead, Syngenta Aron Silverston, Demetra Vlachos and Catherine Kramer – Syngenta RTP Public: Outline ● Herbicide resistance / glyphosate-resistant weeds ● Soybean herbicide market overview and perspective ● Why herbicide tolerant soybeans ● Herbicide tolerant soybean development ● Regulatory and product safety considerations ● New herbicide tolerant trait technology on the horizon 2 Public Weed resistance to numerous herbicides is expanding globally Heap, I. The International Survey of Herbicide Resistant 3 Public : Weeds. Online. Internet. Friday, January 10, 2014 . 4 Public Glyphosate resistance continues to spread in North America http://www.resistancefighter.com/news.aspx 5 Public Growers reporting glyphosate resistance continues to increase Source: 2013 Syngenta Proprietary Research 6 Public: Confirmed glyphosate resistant acres infested Source: 2013 Syngenta Proprietary Research 7 Public: Growth of soybean Pre + Post selective herbicide market Acres (M) Grower Expenditures (M) 50 Pre Acres 500 Pre $ 45 Post Acres 450 Post $ 40 400 35 350 30 300 25 250 20 200 15 150 10 100 5 50 0 0 2009 2010 2011 2012 2013 2009 2010 2011 2012 2013 2009 2010 2011 2012 2013 2009 2010 2011 2012 2013 % of Pre + Post Selective Acres Treated % of Pre + Post Selective Grower Expenditures Post Acres 49.5% 44.8% 37.5% 41.5% 41.5% Post $ 41.1% 38.8% 30.8% 29.8% 32.8% Pre Acres 50.5% 55.2% 62.5% 58.5% 58.5% Pre $ 58.9% 61.2% 69.2% 70.2% 67.2% Source: 2013 Syngenta Proprietary Research 8 Public : % Change of expenditures in weed control - 2009 to 2013 2013 total grower expenditures increased by $266,474,663 (+22.4%) 2013 = $1,457,626,930 2012 = $1,191,152,267 Source: 2013 Syngenta Proprietary Research 9 Public : Limited diversity in useful selective soybean herbicides today All selective soybean herbicides Herbicide Site of Action & Group quizalofop sethoxydim ACCase Inhibitors 1 clethodim fluazifop chlorimuron Selective soybean herbicides chloransulam with POST broadleaf activity flumetsulam thifensulfuron ALS Inhibitors 2 Herbicide Site of Action & Group Selective soybean imazaquin chlorimuron herbicides with activity imazethapyr chloransulam imazamox against Amaranthus flumetsulam metribuzin 5 Photosystem II Herbicide Site of Action & Group bentazon 6 thifensulfuron ALS Inhibitors 2 Inhibitors linuron 7 imazaquin lactofen clomazone Diterpene Synthesis Inhibitors 13 imazethapyr acifluorfen PPO Inhibitors 14 POST fomesafen lactofen imazamox acifluorfen bentazon Photosystem II Inhibitors 6 fomesafen flumiclorac lactofen PPO Inhibitors 14 metribuzin Photosystem II 5 flumioxazin acifluorfen linuron Inhibitors 7 sulfentrazone fomesafen PPO Inhibitors 14 fluthiacet fomesafen flumiclorac saflufenacil flumioxazin PPO Inhibitors 14 pendimethalin fluthiacet sulfentrazone ethalfluralin Microtubule Inhibitors 3 PREM acetochlor trifluralin s-metolachlor Long-Chain Fatty 15 acetochlor dimethenamid Acid Inhibition s-metolachlor Long-Chain Fatty Acid pyroxasulfone alachlor Inhibition 15 dimethenamid pyroxasulfone 10 Public: Herbicides provide tremendous value to agriculture ● The Crop Protection Research Institute calculated that it would take 70 million workers to hand weed all the commercial crops in the U.S. in a single year - Delta Farm Press – October 29, 2013 http://m.deltafarmpress.com/management/how-many-workers-would-be-needed-hand-weed-us- crops?utm_content=buffer2d6bb&utm_source=buffer&utm_medium=twitter&utm_campaign=Buffer 11 Public: Herbicide tolerant crop development Gene discovery is the first step to developing herbicide tolerant soybean technology ● Genes for herbicide tolerance can come from varied sources - Plants - Bacteria ● Candidate gene mode of action tested biochemically ● Validated in transgenic plants ● Herbicide tolerance can be delivered by various methods - Metabolism - Overexpression - Tolerant enzyme or target site - Changes in binding efficiency of enzyme targeted by the herbicide 13 Public Examples of herbicide tolerance genes Gene Herbicide Source Mechanism of Tolerance Tolerance CP4 (EPSPS) glyphosate soil bacterium EPSPS enzyme tolerant to Agrobacterium sp. inhibition by glyphosate strain CP4 pat glufosinate soil bacterium Enzyme which inactivates Streptomyces glufosinate viridochromogenes avhppd-03 mesotrione, oat plant Lower binding affinity to HPPD- isoxaflutole (Avena sativa) inhibiting herbicides DMO dicamba bacterium Enzyme which demethylates Stenotrophomonas dicamba maltophilia 14 Public: A typical single-event contains two transgenes – the gene of interest and a marker for selection of transformed plants Transferred DNA (genes and regulatory elements) Plasmid sequence Plasmid sequence Selection Promoter 1 Gene of Interest Promoter 2 Marker • Gene of interest is inserted with other genetic elements in a “cassette” to produce the desired effect (e.g., herbicide tolerance) • “cassette” design affects the performance of the gene of interest • The goal is to express the protein of interest at the right time and in the appropriate plant tissue • Expression cassettes can contain following elements: • Gene of interest – encodes for production of the protein that provides tolerance • Promoter – directs how much protein should be produced or “translated” from the gene of interest • Enhancer – enhances the translation to increase the “expression” or tolerance • Selectable marker – gene that confers a trait that allows identification during transformation of cells that carry the new trait • Terminator sequence – stop signal to end the translation 15 Classification:Public: PUBLIC Agrobacterium Transformation Agrobacterium is a natural genetic engineer It has the ability to insert its own DNA into a plant cell Integration New Gene Infection Agrobacterium tumefaciens Plant Cell Transferred DNA (genes and regulatory elements) Plasmid sequence Plasmid sequence Selection Promoter 1 Gene of Interest Promoter 2 Marker 16 Classification:Public: PUBLIC Transformation is the process used to insert genes of interest into the target plant ● A single cell gets the new gene and is regenerated into a plant. ● Each individual plantlet that survives the selection during transformation is considered an “event” ● During transformation it is desirable to create as many events as possible to increase chance of success - Target creation of hundreds or even thousands of events for a herbicide tolerant product ● Having more events gives a better chance at finding and developing an event with necessary desirable characteristics Hans J. Bohnert, University of Illinois and Henry T. Nguyen, University of Missouri-Columbia 17 Public: Characteristics of a Commercializable Event ● Single Copy ● No extra DNA ● No changes in sequence ● Soybean agronomics unchanged - Yield, maturity, height, etc. ● Performance of trait matches commercial need (e.g., 4x Herbicide treatment V3) 18 Public: How to create a transgenic soy event 19 Public: Event testing and selection for the best event to commercialize ● Glasshouse selection for herbicide tolerance to screen out weak events - Requires small number of seed and can be done on a more high- throughput basis ● Early evaluation of events to determine if any should be excluded due to the quality of the insert into the soybean genome 20 Public: Event testing and selection for the best event to commercialize ● Field selection can occur once sufficient seed quantities exist 1) Herbicide efficacy to select events with acceptable crop tolerance • Up to several dozen events at multiple locations • Herbicide rates as needed, e.g., 4X 2) Soybean yield and agronomic performance • Usually a more limited number of events for one or more seasons in North and South America • As many as 20 locations per season • Herbicide rates as needed, e.g., 4X 3) Performance in a limited number of representative elite genetic backgrounds Non-transformed Herbicide tolerant Event selection trial - Iowa 21 Public: New soybean variety development begins with trait introgression ● Once an event is selected for commercialization, introgression to create new soybean varieties begins - A donor plant carrying the herbicide tolerance trait of interest is crossed with elite soybean lines - Progeny are back-crossed to the parental line and then selfed to “fix” the trait while recovering the elite parents’ genome . - Marker-assisted and conventional breeding techniques are used to create elite soybean lines containing the new herbicide tolerance event ● The goal is to create elite soybean lines with: - High yield potential - Favorable agronomic package - New herbicide tolerance trait 22 Public: Herbicide tolerant soybean lines are selected and tested for performance before becoming a new commercial variety ● A new line will start with the selection of an individual plant - Based on phenotypic and genotypic characteristics ● Single plant selection will become a progeny row which is compared to commercial standards for performance, etc ● New lines are selected for advancement to a commercial track based on performance and agronomic traits ● After sufficient seed quantities exist, new lines enter the pool of candidates to become new commercial varieties - Subjected to multiple years of multi-location yield testing and characterization 23 Public: Product safety requirements ● Numerous studies are conducted to evaluate the safety of a new herbicide tolerant event ● Molecular characterization of the genetic event ● Protein Safety to ensure there are no potential