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 risks to humans or livestock - Toxicity of protein - Allergenic potential of protein - Digestibility of the protein - Stability of protein ● Protein expression study - Produce an expression profile in the crop to provide data for exposure assessments

24 Public: Product safety requirements

● Compositional Assessment - Field trials at multiple locations - Forage and grain (seed) samples collected and analyzed for >70 soybean nutritional components - Analyzed for similarity to non-transformed control as well as a number of non-transgenic commercial reference varieties ● Nutritional Assessment based on compositional assessment and analysis - Animal Feeding studies to confirm no adverse affects from using feed containing meal from transgenic soybeans ● Takes 3-4 years to complete required product safety work and the dossiers - More than 25 comprehensive studies needed for global submission of a single event for US cultivation approval and import approval in key countries

25 Public: Compositional assessment examines more than 70 nutritional components

26 Public: Phenotypic and agronomic characteristics are carefully examined Phenotypic characterization of a new herbicide tolerant crop Characteristic Variable tested Test location Germination, dormancy, emergence Seed germination and dormancy Growth Chamber Seed germination and emergence Field Vegetative growth Seedling vigor Field Plant height Field Lodging Field Reproductive growth Days to 50% flowering Field Pollen viability Field Pollen morphology Greenhouse Flower color Field Days to maturity Field Seed test weight Field Seed moisture Field Grain yield Field Plant survival Final stand count Field Seed dispersal Pod shattering Field Ecological interactions Insect feeding & disease presence Field

27 Public: Completion of the safety and regulatory phase

● Requires 50+ combined studies and 6+ years to conduct the studies and obtain regulatory approvals ● Safety data packages needed to gain regulatory approvals for: - cultivation and use in countries where the crop will be grown - import approvals for use as food and feed in key soybean export markets (multiple agencies across 15 or more countries) ● Regulatory approvals give technology providers a license to sell and help to insure growers have access to the global markets for their crop

28 Public: SYHT0H2 development followed this process

● Transformation in 2007

● First events field tested in North America during 2008

● SYHT0H2 was selected as the lead event in 2011 after testing in North & South America across five field seasons

● Efficacy and agronomic performance confirmed in elite genetic backgrounds across a broad range of maturity groups and environments in North & South America during 2012 and 2013

MGI herbicide tolerant soybeans are not approved for 29 Public: use in the U.S. and are not being offered for sale. Herbicide tolerance & agronomic evaluation

Herbicide tolerance evaluations Agronomic evaluations ● Multiple locations per genetic line ● 876 genetic lines tested - MG0 to MGVI ● Multiple locations (7 to 10) per ● Herbicide tolerance data genetic line collected at regular intervals - MG0 to MGVI ● Agronomic data and soybean yield

Rate Additives Growth Treatment (g ai/ha) (v/v) Stage mesotrione (4X) 840 None PREM isoxaflutole (4X) 420 None PREM mesotrione (4X) 420 2.5% AMS + 0.25% NIS V2-3 Isoxaflutole (4X) 280 2.5% AMS + 0.25% NIS V2-3 glufosinate sequential (2X / 2X) 900 to 1185 2.5% AMS V2-3 / V6

MGI herbicide tolerant soybeans are not approved for 30 Public: use in the U.S. and are not being offered for sale. Agronomic performance in diverse genetics 4X rates post-emergence

110 ● No yield difference between 100 sprayed and unsprayed 90 soybeans 80 ● No effect on soybean 70 maturity or plant height 60 50 40 30 20 Yield (% of (% Untreated) Yield 10 0 mesotrione isoxaflutole glufosinate 420 g 280 g 1185 g

Number of genetic lines tested for each herbicide mesotrione = 108; isoxaflutole = 35; glufosinate = 133

MGI herbicide tolerant soybeans are not approved for 31 Public: use in the U.S. and are not being offered for sale. Tolerance to mesotrione and isoxaflutole applied at V2-3 (Ames, IA – 7, 13 & 32 DAT)

Mesotrione 420 g ai/h (4X)

Isoxaflutole 280 g ai/h (4X) MGI herbicide tolerant soybeans are not approved for 32 Public: use in the U.S. and are not being offered for sale. MGI herbicide tolerant soybeans summary

● A new herbicide tolerant soybean technology from Syngenta and Bayer CropScience tolerant to Mesotrione, Glufosinate & Isoxaflutole (MGI) pending regulatory approval

● MGI herbicide tolerance will enable soybean growers to use two modes of action for weed management that provides: - New tool to deliver best-in-class residual control - New options to effectively manage resistant weeds

Key Grower Benefits:

• Enable more herbicide chemistry options in soybeans Untreated • Bring proven residual control • Excellent compatibility with other herbicide tolerant traits and other soybean herbicides • Broad-spectrum dicot and grass control - Including control of resistant weed biotypes • Better, more sustainable weed management

Mesotrione-based Program

Forward looking statements subject to change MGI herbicide tolerant soybeans are not approved for 33 Public use in the U.S. and are not being offered for sale. Increased diversity in soybean herbicides with herbicide tolerant crops Herbicides with activity against Amaranthus with herbicide tolerant crops Herbicide Site of Action & Group Selective soybean 2,4-D Growth Regulators 4 herbicides with activity dicamba glufosinate Glutamine Synthesis

against Amaranthus Inhibitor 10

Herbicide Site of Action & Group lactofen POST lactofen acifluorfen PPO Inhibitors 14 acifluorfen PPO Inhibitors 14 fomesafen POST fomesafen mesotrione HPPD Inhibitors 27 isoxaflutole

metribuzin Photosystem II 5

linuron Inhibitors 7 metribuzin Photosystem II 5 fomesafen

Inhibitors flumioxazin PPO Inhibitors 14 linuron 7 sulfentrazone fomesafen PREM acetochlor flumioxazin PPO Inhibitors 14

s-metolachlor Long-Chain Fatty 15 sulfentrazone dimethenamid Acid Inhibition acetochlor

pyroxasulfone PREM s-metolachlor Long-Chain Fatty 15 dimethenamid Acid Inhibition pyroxasulfone mesotrione HPPD Inhibitors 27 isoxaflutole

34 Public: Conclusions

● Herbicide tolerant soybeans are effective tools for weed management - Effective way to enable new modes of action into soybeans ● Significant time and investment is required to bring these products to the market - Up to 10 years from initial transformation to market - Years of product safety testing and regulatory work - Years of product development and field testing ● MGI herbicide tolerant soybean are one example of a new herbicide tolerant trait technology for soybean

MGI herbicide tolerant soybeans are not approved for 35 Public: use in the U.S. and are not being offered for sale.