Seed Soak Bioassay for Identifying the Roundup Readytm Gene in Sugar Beet Seed

Iowa State University Capstones, Theses and Creative Components Dissertations

Fall 2020

Seed soak bioassay for identifying the Roundup ReadyTM gene in sugar beet seed

James Terry

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Recommended Citation Terry, James, "Seed soak bioassay for identifying the Roundup ReadyTM gene in sugar beet seed" (2020). Creative Components. 684. https://lib.dr.iastate.edu/creativecomponents/684

This Creative Component is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Creative Components by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Seed soak bioassay for identifying the Roundup Ready™ gene in sugar beet seed

James Terry

A creative component submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Seed Technology and Business

Program of Study Committee: Susana Goggi, Major Professor Jacquelyn Ulmer, Committee Member

Iowa State University Ames, Iowa 2020

TABLE OF CONTENTS

Page

LIST OF FIGURES ...... iii

LIST OF TABLES ...... v

NOMENCLATURE ...... vi

ACKNOWLEDGEMENTS ...... vii

ABSTRACT ...... 1

CHAPTER Ⅰ INTRODUCTION ...... 3

Roundup Ready™ sugar beets ...... 3 Roundup™ herbicides ...... 4 Economics of Roundup Ready™ technology in sugar beets ...... 7

CHAPTER Ⅱ LITERATUE REVIEW ...... 9

Quality control and biotech trait testing ...... 9 Herbicide bioassays ...... 12

CHAPTER Ⅲ MATERIALS AND METHODS ...... 19

Sugar beet seed soak bioassay ...... 19 Planting ...... 23 Seedling evaluation ...... 24 Statistical methodology ...... 24

CHAPTER Ⅳ RESULTS AND DISCUSSION ...... 26

Results ...... 26 Calculations for percent Roundup Ready™ ...... 32 Pearson’s Chi-square statistic ...... 33 Discussion ...... 34

CHAPTER Ⅴ CONCLUSION ...... 37

REFERENCES ...... 39 iii

LIST OF FIGURES

Page

Figure 1 Sugar beet spray test performed in greenhouse on HIL2238NT ...... 14

Figure 2 Sugar beet seed soak in 2% solution of Roundup™ Power Max ...... 16

Figure 3 Two commercial seed lots from Hilleshög Seeds ...... 19

Figure 4A Conventional seed lot tested for Roundup Ready™ trait using lateral Flow strip ...... 20

Figure 4B Lateral flow strip with positive result from Roundup Ready™ seed lot ...... 20

Figure 5 Sugar beet seeds are precisely counted to known proportions ...... 21

Figure 6 Lab station used for mixing herbicide solution ...... 22

Figure 7A Vacuum planter head for 100 seeds ...... 23

Figure 7B Pleated filter paper used as germination medium ...... 23

Figure 8 Watering PP with 40 ml of dyed green filtered water ...... 24

Figure 9 Germination chamber set at 20℃ ...... 24

Figure 10A Evaluation of 100% Roundup Ready™ seed lot at 7 days ...... 26

Figure 10B Evaluation of 100% conventional seed lot at 7 days ...... 26

Figure 11 Susceptible sugar beet seedling showing severe toxicity symptoms ... 27

Figure 12 Abnormal seedling vs. susceptible seedling ...... 27

Figure 13A Roundup Ready™ water control at 7 days ...... 28

Figure 13B 100% Roundup Ready™ seed lot with herbicide soak at 7 days ...... 28

Figure 13C Conventional water control at 7 days ...... 28

Figure 13D 100% conventional seed lot with herbicide soak at 7 days ...... 28 iv

Figure 14 Display photo of replication 1 at 7 days ...... 29

Figure 15 Display photo of replication 2 at 7 days ...... 29

Figure 16 Display photo of replication 3 at 7 days ...... 30

Figure 17 Display photo of replication 4 at 7 days ...... 30

Figure 18 Roundup Ready™ seedling with no symptoms next to susceptible seedling with toxicity symptoms at 7 days ...... 31

LIST OF TABLES

Page

Table 1 Percent RR sugar beet seeds in five seed lots tested using seed soak bioassay method ...... 32

Table 2 Chi-square for each seed lot with observed value being the tolerant seedlings, excluding the 100%RR and 100%S seed lots from Table 1 ...... 33 vi

NOMENCLATURE

A.I. Active Ingredient

AOSA Association of Official Seed Analysts

AOSCA Association of Official Seed Certifying Agencies

AP Adventitious Presence

ELISA Enzyme-Linked Immunosorbent Assay

FSA Federal Seed Act

GMO Genetically Modified Organism

HT Herbicide Tolerant

MSDS Material Safety Data Sheet

PCR Polymerase Chain Reaction

PP Pleated Filter Paper

PPE Personal Protective Equipment

RR Roundup Ready™

S Susceptible

SGT Standard Germination Test

QC Quality Control

vii

ACKNOWLEDGEMENTS

I would like to thank my committee chair, Dr. Susana Goggi, and my committee member, Jacquelyn Ulmer, for their guidance and support throughout the course of this research. I would also like to thank Lori Youngberg for her excellent coordination efforts in the Seed Technology and Business graduate program.

In addition, I would also like to thank my friends, colleagues, the department faculty and staff for making my time at Iowa State University a wonderful experience. I want to offer my appreciation to Hilleshög Seeds LLC and their laboratory technician,

Deborah Guilkey, for allowing me to do my research in their laboratory.

Finally, I want to thank my family for their encouragement and my wife for her hours of patience, sacrifice, support and love.

ABSTRACT

Herbicide bioassay methods that involve paper media are successfully used in crops like soybeans, corn, canola and cotton. The spray bioassay method is the current industry standard for herbicide trait testing in commercial sugar beets. The duration of this spray bioassay takes 30 or more days to complete, depending on greenhouse conditions. Herbicide bioassays are an important tool to determine the presence or level of herbicide traits in a seed lot. The timeliness of receiving herbicide trait purity data on a seed lot is essential for the production, manufacturing and marketing of sugar beet seed. Based on different herbicide bioassays used in the seed industry for other crops, a seed soak bioassay could be an ideal candidate for shortening bioassay duration in sugar beets. The objective of this experiment is to evaluate the accuracy, reliability and repeatability of a seed soak bioassay in assessing herbicide tolerance on sugar beets in 7 days. The seed lots used for this experiment included one susceptible (S) sugar beet seed lot and one known Roundup Ready™ (RR) sugar beet seed lot. Seeds from these two seed lots were mixed into five known ratios of herbicide tolerance and susceptibility, to create five new seed lots. The ratios used were 100% RR, 75 RR:25 S , 50 RR:50 S, 25

RR:75 S and 100% S. Four replications of each ratio were immersed for 24 hours in a

2% solution of Roundup™ Power Max formulation [48.7% active ingredient (a.i.)], for a concentration of 0.974% a.i. After 24 hours of submersion, seeds were removed from the herbicide solution, planted in pleated filter paper (PP) and moistened with water.

Bioassays were placed inside a growth chamber set at a constant 20°C, with a 2

photoperiod of 8hr light: 16hr dark, for 7 days. RR and S check samples were planted together with each replication. These check samples were exposed to the same herbicide treatments as the test samples. Water checks were also used as a comparison of normal and abnormal seedlings, as well as any growth rate symptoms. Susceptible seedlings showed severe toxicity symptoms of yellow to brown in color and extreme stunting of the hypocotyl-radicle area. Seedlings with the RR gene had normal growth and development. The results from this experiment indicated that a seed soak bioassay can be used to accurately, reliably, and quantitatively determine herbicide tolerance in sugar beet seed lots. Moreover, the seed soak bioassay can be completed in 7 days compared to the lengthier spray bioassay.

CHAPTER Ⅰ

INTRODUCTION

Roundup Ready™ sugar beets

Sugar beet (Beta vulgaris L.) is an important source of domestically produced sugar in temperate regions of the world (Harveson, 2020). While most of the world’s sugar beet production is conventional, the United States and Canada rapidly adopted the newly introduced GMO sugar beets by 2008 (Zicari et al., 2019). The biotechnology trait used to develop GMO sugar beets is an herbicide tolerant (HT) trait called Roundup

Ready™ (RR). The RR trait, when inserted into a plant’s genome, gives the plant tolerance to the widely used herbicide glyphosate (Mannerlöf et al., 1997). Glyphosate- resistant sugar beets were developed by Monsanto and KWS by early 2000’s and were deregulated by the USDA in 2005 (USDA, 2020). RR Sugar beets became widely available for commercial use by the 2008 growing season (Spieker, 2017). The adoption of new crop improvement tools, such as biotechnology, is essential for the agriculture industry to meet current and projected global demands for food, feed, fiber and fuel.

Since the introduction of HT traits in sugar beets, quality control (QC) and trait purity testing have become critically important.

Sugar beet is an epigeal dicot and part of the Amaranthaceae family (Zicari et al.,

2019). The RR gene was introduced into sugar beets using the agrobacterium transformation method. The Agrobacterium tumefaciens (strain CP4) is a soil bacterium that naturally produces the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase 4

(EPSPS) (Gutormson et al., 2005). The EPSPS enzyme participates in the biosynthesis of amino acids and is essential to the bacterium’s metabolic biochemical pathways

(Steinrucken & Amrheim, 1980). Glyphosate-resistant sugar beet inbred lines are transformed using the CP4 EPSPS gene from Agrobacterium sp., to create highly tolerant cultivars (Mannerlöf et al., 1997). The agrobacterium is used as a vector to infect the sugar beets cells where DNA is transferred and reproduced (Tunning & Stahr,

2019). Although this process takes time, it is more likely to transfer a single gene copy compared to the particle bombardment method (Tunning & Stahr, 2019). Many independent transformations were evaluated, to find an event with the greatest efficacy.

After years of research and development, event H7-1 was selected as commercially viable and was registered through the regulatory approval process in the United States and Canada. Since commercial deregulation, RR sugar beet varieties sold by Hilleshög

Seeds LLC use event H7-1. Thus, RR sugar beets are available for producers throughout the growing regions of the United States and Canada.

Roundup™ herbicides

Glyphosate has become one of the most widely used herbicides in modern agriculture. It is an easy and flexible tool to control weeds in many different settings and it helps producers be more efficient and productive with their crops (EPA, 2019).

Glyphosate is both environmentally and economically preferable over conventional agronomic practices (EPA, 2019). Studies have shown that RR sugar beet’s response to glyphosate causes no damage to the crop and root yield tends to increase as the number 5

of glyphosate applications increase from one to two (Mesbah & Miller, 2004). Some advantages of glyphosate over conventional herbicides include safer environmental toxicology, improved crop safety, flexibility in timing of application, controls larger weeds, has a broader weed spectrum and capabilities of controlling weeds that have become resistant to herbicides with alternative modes of action (Guza et al., 2002).

Glyphosate is typically applied twice in a growing season, once when the seedling reaches the first true leaf stage and the second between the 8 and 12 leaf stage, prior to canopy closure. This allows for full weed control throughout the growing season. Using glyphosate-resistant sugar beets and Roundup™ herbicides reduces the number of trips through the field, therefore, decreasing chemistry costs, fuel, labor and soil compaction

(Guza et al., 2002). Most sugar beet farmers also grow other RR crops, which helps standardize on farm chemical regimes. Glyphosate has helped farmers adopt reduced tillage and no-till practices, which reduce soil erosion and has other positive effects on the environment such as improved water quality (Cerny et al., 2010).

The a.i. in Roundup™ herbicide formulations is glyphosate, which is the common name for N-phosphonomethyl-glycine. Roundup™ is a group 9 herbicide with a mode of action as an amino acid inhibitor (Ross & Childs, n.d.). Glyphosate is a systemic herbicide that is taken up by the foliage and translocated throughout the plant to build up in the meristematic zones (Mannerlöf et al., 1997). Glyphosate binds to the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and blocks its activity in the shikimate pathway, inhibiting the biosynthesis of a critical amino acids (Steinrucken

& Amrheim, 1980). When Roundup™ herbicide is used with RR crops, containing the 6

CP4 EPSPS transgene, the herbicide has selective broad-spectrum weed control.

Commercially available RR crops include soybeans, corn, cotton, canola, sugar beet, bent grass, and alfalfa, with many more crops under development (Fernandez-Cornejo et al., 2014). When the herbicide is not used in a glyphosate-resistant crop, it acts as a non- selective broad spectrum weed control and kills all plants not containing the transgene.

Glyphosate can also be used cautiously as a desiccant to kill off foliage, allowing a crop to dry down faster for an earlier harvest. It is important to note that glyphosate can be translocated to the seed and destroy the seed lot when the compound is used as a crop desiccant.

Prolonged and overuse of glyphosate has created some issues with weed resistance. Herbicide resistance in weeds is not novel or unique to glyphosate. It has become an issue with many chemistries and modes of actions. In 2018, thirty-eight weed species worldwide had developed glyphosate resistance (Heap & Duke, 2018). There are reports of additional resistant weed species throughout the world, but more confirmation is required. Herbicide tolerance can occur naturally, by selective pressure from single- herbicide overuse, by natural or induced mutations or by genetic modification using a specific gene (SCST, 2016). It has become inevitable that the list of glyphosate-resistant weeds will continue to grow as the herbicide continues to be used without implementing appropriate resistance-management practices. Moreover, Roundup™ herbicide can be harmful to humans and has been determined to be a carcinogen in the state of California.

This development has placed the manufacturers of the herbicide, Bayer and its Monsanto subsidiary, under severe scrutiny; and their herbicide-safety claims have been challenged 7

in court. It has become clear through decades of use that the advantages of using

Roundup™ herbicides outweigh the limitations if used with appropriate resistant- management and safety measures.

Economics of Roundup Ready™ technology in sugar beets

HT traits have revolutionized the way farmers grow sugar beets in North

America. The adoption rate of RR sugar beets in the U.S. has been more rapid than any other biotech crop, with over 95% of hectares planted in less than two years after commercialization (Western Sugar, 2015). This rapid adoption rate demonstrated the crucial need, by North American farmers, for biotechnology in high value crops like sugar beets. Weeds in sugar beet fields pose a major problem for farmers by competing for the plant’s resources. Optimal weed control programs give the producer greater economic return through greater root and greater extractable sucrose yields. Prior to the introduction of RR sugar beets, weed management was one of the largest expenditures for sugar beet farmers (Odero et al., 2008). Sugar beet producers would use a combination of conventional herbicides, cultivation and hand labor to control weeds in fields. A conventional herbicide program consisted of 2-3 applications of 2-5 different a.i.’s costing in the wide range of $57 to $393/ ha, while a glyphosate-resistant management program costs between $40 and $69/ha, depending on rates and number of applications (Kniss, 2010). In 2007, a comparison and economic study of conventional and glyphosate-resistant sugar beets was conducted side by side in a Wyoming producers’ field. The study demonstrated that glyphosate-resistant sugar beets improved 8

net economic return by $576/ha in comparison to conventional practices (Kniss, 2010).

This study is only representative of a single year of production in one farmer’s field and was unable to be duplicated because of the remarkably high adoption rate by 2008.

Although these figures may vary by region and agronomic practices, it still demonstrates the apparent and clear demand for HT traits and biotechnology in sugar beets.

CHAPTER Ⅱ

LITERATURE REVIEW

Quality control and biotech trait testing

Seed lots need to be tested for internal decision making and to provide a basis for marketing and sales of seed products. The Federal Seed Act (FSA) regulates the interstate movement of seed products (Stahr, 2019). The FSA requires that seed transported through interstate commerce have standards for labeling including using truthful information that allows the buyers to make informed decisions. This regulation also applies to the marketing and advertising of seed products. The FSA helps promote fair competition within the U.S. seed trade and consistency among state laws. Seed quality laboratories maintain compliance through seed agencies including the

Association of Official Seed Analysts (AOSA) and the Association of Official Seed

Certifying Agencies (AOSCA). Seed analysts must adhere to the AOSA testing rules and seedling evaluation standards, in states that require these rulings. AOSCA regulates and standardizes official seed certification agencies and establishes standards for genetic purity. The U.S. has some of the strongest seed laws in the world, which helps give consumers confidence in the products they are purchasing, even though most of the seed sold within the U.S. is not certified. Seed sold internationally, such as seed going to

Canada, requires certification.

Quality testing is at the core of any seed business. Biotechnology and genetically engineered crops have created a need for additional QC methods not necessary in 10

conventional seed products. These QC methods determine the trait purity of a seed lot and are also used to detect adventitious presence (AP) in the seed or grain value chain.

AP refers to low levels of unintended material, such as biotech traits in seed, grain or feed and food products (USDA, n.d.). AP occurs when biotech traits are present in a conventional variety and can occur in a seed lot from improper coexistence or comingling. Contaminates can come from pollen movement during seed production, inadequate cleaning of harvest, transportation, storage, and/or conditioning equipment, or improper blending of seed. Biotech trait purity differs from AP in that the seed analyst is quantitatively determining whether desired traits are present at certain levels.

Whereas, AP testing determines if unintended traits are present and a qualitative method may be sufficient for determining low-level presence (Stahr, 2016). The importance of biotech trait purity testing is to protect both the farmers investment in his crop as well as the seed company. Due to public concern with the safety of GM seed products, issues with food labeling and government regulations QC is becoming increasingly important.

QC helps protect research investments, maintain compliance with government regulations and ensure customer satisfaction (Mumm &Walters, 2001).

QC is conducted to assure that farmers receive the highest quality seed and with desired traits. A good biotech trait purity test should give the technician enough data to accurately determine the trait purity percentage for the seed lot. By law, the seed lot must have a minimum of 95% genetic purity according to the cultivar described on the label. In the sugar beet industry, genetic purity testing includes the RR-purity spray test in addition to counts from red beet and swiss chard grow-outs. For a commercial seed lot 11

to be deemed marketable, it must contain less than 5% red beet, swiss chard and non-RR trait seed contaminants. Without adequate biotech trait testing throughout seed development, a seed company risks making poor decisions on the advancement and commercialization of products. This inadequate genetic purity can cause delayed timing to market, obsolete marketing efforts and potential loss of revenue if defects are found after substantial conditioning steps have taken place, or if a seed lot must be recalled. A seed company could lose market share based on all these factors. Biotech testing is also critically important to assess for AP. QC relies on capturing accurate data in a timely manner to be used in decision making and to give all stakeholders quality assurance.

There are three main methods used in herbicide trait purity testing, which are bioassay, immunoassay, and polymerase chain reaction (PCR). There are different capabilities of each testing method. Herbicide bioassays are designed to subject seed or plants to an herbicide solution for determining the tolerance to the herbicide and the presence of HT traits. Herbicide bioassay methods can be in the form of a seedling spray, substrate imbibition, seed soak or a combination of these methods. Immunoassay or Enzyme-Linked Immunosorbent Assay (ELISA) tests, determine the presence of a trait by identifying the known transgenic protein in seeds or plants. There are two types of immunoassays, lateral flow strips or plate tests. PCR is used to test seed or plants for the presence of the specific DNA sequence for the HT trait. PCR testing is also used in

AP testing. In this case, PCR is used to screen for the presence of promoter and terminator sequences as well as the specific herbicide trait sequence. A promoter and terminator sequence are components of the transgene, where the promoter allows the 12

transgene to express in the plant and the terminator sequence stops the protein translation, so the transgene codes the correct protein sequence and structure (Tunning &

Stahr, 2019). This method can be used to ensure no unintended events or competitor’s traits have contaminated a seed lot. Whereas PCR checks for the specific DNA sequence and ELISA locates the specific protein encoded by the transgenic DNA, bioassays indirectly determine the transgenic activity by evaluating the effect of the gene in a living plant (SCST, 2016). There are advantages and limitations to each of these testing methods and the proper test or combination of tests should be decided depending on the laboratory’s capabilities and data requirements.

Herbicide bioassays

Herbicide bioassays are a simple and inexpensive way of determining the presence of an HT trait like the RR gene. When developing a program to screen, evaluate and monitor herbicide resistance, the most important criteria is the selection of a herbicide bioassay to use (Beckie et al., 2000). All bioassay methods for testing biotech trait purity involve the germination, growth and development of seedlings. The seed must be imbibed as the first step in the germination process. The seed is hydrated, and the activation of enzymes and mobilization of food reserves begins. Hydrolytic enzymes break down stored reserves of starch, lipids and proteins into simpler forms of sucrose, amino acids and fatty acids to be used as building blocks for synthesis of new material required for cell division and cell expansion. The embryo then initiates growth while the endosperm is weakened by enzymatic activity and from the breakdown of food 13

reserves (Arora, 2017). The radicle ruptures the seed coat, protrudes and elongates pulling the hypocotyl and shoot from the fruit wall. Seedling emergence has thus occurred and can be evaluated.

There are many different factors to consider when adopting a biotech trait purity evaluation method. Some influences include how quickly the seed company needs the data, whether these bioassays will be used for preliminary decision making, or if a molecular approach is desirable for a complete genotype identification. In the case for sugar beets, most molecular testing for trait purity takes place at the pre-basic and basic seed level, before a commercial crop is produced. A spray test is then used for the commercial seed lot. Other important factors in any laboratory environment is the experience and knowledge of the technician who is conducting, reading and interpreting the bioassay. Labor requirements, equipment and space needed for testing, automation potential, germination media and the reliable interpretation of abnormal versus susceptible seedlings are also important considerations. Important herbicide elements to consider are the herbicide concentration, application method, workers exposure during the test, as well as proper disposal of supplies contaminated with the herbicide.

Methodologies using substrate moistened with herbicide solution and immersion of seeds in herbicide solution are feasible, practical and applicable to seed quality assurance laboratories, and do not require special equipment (Faria de Melo et al., 2013). Herbicide bioassays like a seed soak or substrate imbibition also can give the seed analyst an estimate of the germination percentage of the seed lot (Savoy et al. 2001). When 14

considering a bioassay for HT traits, these are some factors to consider before deciding on a spray-out, substrate imbibition, seed soak or a combination of methods.

The spray method is the most widely used herbicide bioassay employed by sugar beet seed companies. The purpose of this test is to determine the biotech trait purity of

RR sugar beet seed lots. The test is performed prior to the pelleting or coating process.

Seeds are sown in soil, usually with a vacuum planter, and grown in a chamber or greenhouse. At least one set of

S check seed is used per table or seed lot (Figure 1). When the seedlings reach the desired growth, at the 4 true-leaf Figure 1: Sugar beet spray test performed in greenhouse on HIL2238NT. Red stakes indicate negative check. stage, herbicide is sprayed uniformly over the foliage at a similar application rate as in the field. Initial evaluation occurs at 7 days with a final evaluation at 10 days.

Susceptible (non-RR) sugar beet seedlings will show symptoms of yellowing or browning of the shoot structure. New growth on susceptible seedlings can be curled and yellow, or the entire seedling can become dry and die. Evaluating this bioassay can be easier than other methods because there are few abnormal seedlings present during the evaluation. Results are reported to the quality and supply chain managers as a percent of non-RR seedlings. If greater than 5% susceptible plantlets are presented, the seed lot is removed from the market, purchase orders are halted and substitute seed lots are found. 15

If less than 5% susceptible plantlets are presented, then the seed lot is considered marketable, purchase orders are approved, and the seed lot is pelleted and treated. The spray bioassay method can be made less labor intensive by automation. The disadvantage that sugar beets and other crops face with this method is the time and space required to perform the test. Sugar beet seedlings can take 2-3 weeks to reach the 4 true- leaf stage. An additional 7-10 days is needed for susceptible seedlings to begin showing symptoms from the Roundup™ application. The total duration of the spray method is 30 or more days, depending on greenhouse conditions. The more rapidly RR-purity data can be generated, the sooner purchase orders can be approved, and manufacturing can continue with conditioning the seed lot for sale.

The substrate imbibition bioassay method involves wetting the germination media with a herbicide solution before the seeds are planted. The herbicide solution for this bioassay is mixed at a lower concentration than for the spray or seed soak methods.

Seedlings with the HT trait must germinate normally inside the growth chamber, imbibing low doses of herbicide, while herbicide-susceptible seedlings must express phytotoxicity symptoms. If the herbicide concentration is too high, it could cause HT seedlings also to express symptoms of phytotoxicity. Substrate imbibition methods are a cost-effective way to obtain HT trait purity data which encourages the testing of more seeds, giving a greater confidence level in the results (Savoy et al., 2001). Germination medium commonly used for the substrate imbibition bioassay are crepe cellulose paper, towels or blotters. After the herbicide solution is applied to the substrate, the seeds are then planted on the media where they imbibe the solution for the duration of the test. 16

Both S and HT check samples are included with each sample or replicate to corroborate the bioassay’s efficacy. The evaluation of this bioassay can be conducted at 7 days.

When evaluating this bioassay, abnormal seedlings can be detected just as in a standard germination test (SGT). The laboratory technician is exposed to the herbicide during planting, reading and cleanup so personal protective equipment (PPE) is required at all stages of the bioassay. Proper disposal of media soaked in herbicide and cleaning of the equipment dedicated to the herbicide is of critical importance.

A seed soak bioassay involves applying a herbicide treatment to the seeds prior to being planted on the germination medium. One technique is to imbibe the seeds with water in a towel and then a short soak in a diluted herbicide, while another technique

Figure 2: Sugar beet seed soak in 2% solution of Roundup™ Power Max. Seeds are immersed in herbicide solution for 24 hours. involves a pre-treatment of soaking the seed in a diluted herbicide during initial imbibition (Gutormson et al., 2005). (Figure 2). Goggi and Stahr (1997) also developed a successful seed soak method to determine the presence of the RR gene in soybeans using a 2% solution of Roundup™. Todd Gaines, with the Colorado State University Weed

Science Department, developed an unpublished seed soak bioassay method for detecting 17

the AXigen trait in wheat, where the seed is immersed in an Aggressor herbicide solution for 24 hours and then planted in the germination media (Gaines, 2020). When using bioassay methods where seed is immersed in an herbicide solution, the removal of excess herbicide is important to avoid phytotoxicity symptoms in tolerant seedlings

(Gutormson et al., 2005). Seed submersed in a herbicide solution must be strained and rinsed, or blotted dry to remove all excess herbicide, before being planted in a paper media (Gaines, 2020). Seed soak bioassays are conducted in a growth chamber, with the same lighting and temperature requirements as a SGT and evaluated at 7 days. As the embryo begins to germinate and the radicle protrudes from the fruit wall, herbicide is actively translocated throughout the roots and shoots. During the evaluation, the roots and shoots should be visible and tolerant seedlings should appear anatomically normal.

Abnormal and dead seed will be noticeable during the evaluation and should be excluded from the calculations. One advantage to this test is that herbicide exposure to the technician is only during the application of the herbicide and not during the reading.

Herbicide bioassays present some disadvantages. For example, obtaining results from these tests can take anywhere from a few days to a few weeks. If a seed company needs an immediate turnaround in data to make decisions, then other testing methods may be more appropriate. Another challenge to bioassays is personnel training. The seed analyst must evaluate different seed germplasm. These different genotypes can express a variation of symptoms from the herbicide treatment. This variation may obscure seedling characteristics and make more difficult distinguishing susceptible seedlings from abnormal seedlings in some seed lots. Additionally, some seed lots may have dead seeds, 18

hard seeds or seeds that do not germinate within the testing period and their tolerance may not be confirmed by an herbicide bioassay. These issues may be resolved, however, by corroborating the herbicide susceptibility of un-germinated or dead seeds using a lateral flow strip. Another matter to consider when selecting an herbicide bioassay is that some germination media, such as filter paper, may dry out during the test. Dehydration of the media may alter the actual herbicide concentration (Cutulle et al., 2009).

There are also safety factors to consider when selecting an herbicide bioassay.

Herbicides may be harmful to humans, thus adequate training for laboratory personnel is required and MSDS recommendations must be followed. Herbicide handling should also be done in designated workspaces and dedicated equipment should be used to avoid any unintended contamination to other tests or exposure to personnel. Disposal of remaining herbicide and soaked substrate waste must also be considered. All hazardous waste must be properly labeled and appropriately disposed of when the testing is complete.

The experimental hypothesis is that a soak bioassay is a reliable method to assess seed lot RR-purity in sugar beets. The objective of this experiment is to evaluate the accuracy, reliability and repeatability of a seed soak bioassay in assessing herbicide tolerance on sugar beets in 7 days.

CHAPTER Ⅲ

MATERIALS AND METHODS

Sugar beet seed soak bioassay

Two Hilleshög seed lots were used to evaluate the accuracy and reliability of the seed soak bioassay on sugar beets. A known 100% RR-purity hybrid, HIL2238NT, produced in 2019 and sold commercially in 2020 was used in this experiment. A representative composite sample from this hybrid was obtained from Batch 19-097 and subdivided into three working samples. One working sample was used to confirm the

100% RR-purity using the spray bioassay method, another working sample was germinated using the SGT, and the last working sample was used for conducting the seed soak bioassay. A

Hilleshög conventional variety from Europe was used as the S seed lot. This Figure 3: Two commercial seed lots from Hilleshög Seeds. HIL2238NT has a hybrid is unknown, but the known RR-purity percentage of 100%. The conventional seed lot is from Europe and is 100% conventional. seed lot was produced in 2013 and has the material identifier 13005090-90 (Figure 3).

This conventional seed lot is used as the RR susceptible control in all spray tests conducted at Hilleshög Seed quality assurance laboratory. Three working samples were drawn from the composite sample, one for the SGT, another for the seed soak bioassay, 20

and the third sample of 30 grams (around 2,000 seeds) was ground up and a CP4 EPSPS lateral flow strip from Agdia was used to confirm that the seed lot did not contain any seeds with the RR gene before moving forward with the seed soak bioassay (Figure 4A,

B).

Figure 4A: Conventional seed lot tested for presence of RR Figure 4B: Lateral flow strip with positive results from trait using a lateral flow strip from Agdia. The lateral flow RR seed lot. strip shows a single band indicating absence of RR gene in the seed lot. 21

Seeds from the S seed lot and RR seed lot were counted by hand and mixed to form five new seed lots of known ratios (Figure 5). The known RR ratios were 100%,

75%, 50%, 25%, and 0% RR. Four replications of 100 seeds were used in each, SGT test and seed soak bioassay. Each replication was done independently (i.e. new glyphosate solution was mixed for each replication and each replication was planted on the following day of the previous replication’s evaluation). The first replication was done side by side with a SGT for both

RR and S seed lots. Figure 5: Sugar beet seeds are precisely counted to known proportions.

A 2% glyphosate solution was prepared using Roundup™ Power Max formulation (48.7% a.i.), for a concentration of 0.974% a.i. in the working solution. A

2% solution was used for this experiment, as recommended in Goggi and Stahr (1997).

When performing routine seed soak bioassays, a stock solution can be used to give greater precision. The total volume of working solution, for each replication of all ratios, was 500 ml consisting of 10 ml Roundup™ and 490 ml of filtered tap water mixed on a magnetic stir plate, in a 500 ml Erlenmeyer flask (Figure 6). Each new seed lot, of known ratio, was placed in five separately labeled SOLO® UltraClear™ graduated cups.

One hundred ml of the 2% working solution was poured into each cup to completely submerge all seeds. The solution and seeds were swirled until all seeds sank to the 22

bottom of the cup. Seeds were soaked for 24 hours ± 1 hour, at 22℃. Since all new seed lots tested contained a mixture from the two original seed lots, only two water controls were used in each replication, the original 100% RR and 100% S seed lots. The water controls were soaked for a 24-hour period in water prior to planting. These SGT water

Figure 6: Lab station used for mixing herbicide solution. controls were included in each replication as a visual reference for identifying seedling abnormalities other than RR-susceptibility. Seeds were then strained through a screen and rinsed for 10-15 seconds under tap water before being placed on a Kimwipe™ or a paper towel and blotted dry to eliminate any excess herbicide and make it easier to plant with a vacuum planter.

Planting

Seeds were planted into GE Whatman 3014 pleated strips 110mm x 20mm, using a 100-seed vacuum planter. This protocol was comparable to planting sugar beet SGT

(Figure 7A, B). PP is a commonly used SGT medium for sugar beet commercial seed lots, breeder seed, and on both raw and pelleted seed. PP makes SGT preparation and reading easier for sugar beets and helps standardize protocol and materials in the

Figure 7A: Vacuum planter head for 100 seeds. Figure 7B: Pleated filter paper (PP) used as germination medium. laboratory. The PP was transferred into a germination tray with a labeled GE Whatman

0858 110 x 580 mm wrapping strip to contain the pleats inside the tray. The trays were watered using 40 ml of dyed green filtered water, this is the same as the SGT (Figure 8).

Green dye is used to confirm the tray has been watered and the PP is adequately moist.

The labeled germination trays are stacked so each pleat is sealed to maintain moisture.

The herbicide-treated samples were placed in a sealed 2-gallon zip-lock bag to ensure no herbicide fumes reached the untreated seeds. The trays were then placed on a middle shelf in a U.S. Cooler germination chamber set at 20℃ for 7 days (Figure 9). The germination chamber has fluorescent tube lighting set with a photoperiod of 8hr light:16hr dark.

Seedling Evaluation

For this experiment, seedlings were evaluated twice, at 7 days and at 10 days, to compare ease of readability and progression of symptoms in non-trait seedlings. During the evaluation on day 7, a record was taken of normal HT seedlings, normal S seedlings, abnormal seedlings and dead seeds. At day 10 all seeds and seedlings were evaluated again to ensure there was no changes in their categories. All seedling evaluation was conducted in accordance to the AOSA rules (AOSA, 2019).

Figure 8: Watering PP with 40 ml of dyed green filtered Figure 9: Germination chamber set at 20℃. water.

Statistical methodology

The difference between the expected and observed RR frequencies in the seed lots was calculated using Pearson’s Chi-square goodness of fit test. Chi-square is a statistical test used to compare observed data, such as seedling counts, with expected RR 25

frequency data stated in the experimental hypothesis. The chi-square statistic compares and measures the size of any discrepancies between the expected data set and the actual results. The tests were estimated using the CHISQ.DIST.RT formula in Microsoft Excel.

The expected frequencies (75%, 50%, 25%) were compared with the observed frequencies to determine if the bioassay accurately determined RR-purity with 2 degrees of freedom and α = 0.05. The degrees of freedom is the number of categories minus 1.

The alpha level or level of significance is the probability of rejecting the null hypothesis when the null hypothesis is true. This would be committing a Type I error. Results from

Pearson’s Chi-square were used to determine whether to accept or reject the experimental hypothesis, which stated that the seed soak bioassay, is a reliable method to assess seed lot RR-purity for sugar beets.

CHAPTER Ⅳ

RESULTS AND DISCUSSION

Results

Seedlings that contain the RR trait grew normally and did not have or only had slight defects that did not impair seedlings from developing into a normal plant under favorable conditions. A normal seedling had a radicle which protruded from the seed coat and a hypocotyl which elongated pulling the cotyledons from the seed coat. The

Figure 10A: Evaluation of 100% RR seed lot at 7 days. Figure 10B: Evaluation of 100% S seed lot at 7 days. All No seedlings showed signs of toxicity from herbicide. seedlings showed signs of severe toxicity from herbicide. epicotyl did not show any development within the testing time. Primary roots developed normally and some secondary roots also developed during the test (Figure 10A).

Susceptible sugar beet seedlings displayed severe toxicity symptoms, including stunting and discoloration in the roots and shoots (Figure 10B). The hypocotyl-radicle were 27

extremely stunted and a yellow to brownish discoloration was observed throughout the root and shoot structures (Figure 11). A seedling was considered abnormal if there was less than half of the cotyledon tissue remaining or if less than half of the cotyledon tissue was free of necrosis or decay.

Abnormal seedlings had characteristics of stunting, malformed, broken hypocotyl or deep cracks or lesions Figure 11: Susceptible sugar beet seedling showing severe toxicity symptoms to herbicide at 7 days. reaching the conducting tissue.

Seedlings were considered abnormal if the root was missing or had a weak and shortened primary root system.

Other abnormal seedling characteristics included seedlings with one or more Figure 12: On the left is an abnormal seedling with only cotyledons protruding and no radicle or hypocotyl visible at 7 days. Note: even though essential structures missing or the seedling is considered abnormal, cotyledon tissue looks to be tolerant. All abnormal and dead seeds were excluded from calculations. On the right a susceptible seedling. damaged (Figure 12). All dead seeds showed no emergence of roots and shoot. Dead seeds, abnormal or non- determined seedlings were evaluated but excluded from the calculations of RR 28

percentage in the seed lot. The water controls were used for a visual confirmation of normal seedlings, as well as abnormal seedlings (Figures 13A, B, C, D). The germination rates indicated by the SGT was 98.25% for the RR seed lot and 99.5% for the S seed lot.

Figure 13A: RR water control used for comparison at 7 Figure 13B: 100% RR seed lot with seed soak treatment days. in herbicide at 7 days.

Figure 13C: Conventional water control for comparison Figure 13D: 100% conventional seed lot with seed soak at 7 days. treatment in herbicide at 7 days. 29

Figure 14: Display photo of replication 1 at 7 days. RR seeds on left, conventional seeds on right.

Figure 15: Display photo of replication 2 at 7 days. RR seeds on left, conventional seeds on right.

Figure 16: Display photo of replication 3 at 7 days. RR seeds on left, conventional seeds on right.

Figure 17: Display photo of replication 4 at 7 days. RR seeds on left, conventional seeds on right.

The non-RR, susceptible seedlings showed severe toxicity symptoms to the herbicide treatment at 7-days. The susceptible seedlings germinated but were stunted with stubby roots. The hypocotyl emerged fully in most cases to expose the cotyledons.

The shoot structures were yellow to brownish in color and necrosis developed from the shoots to the radicle. The susceptible seedlings were about 70% shorter, from shoot to radicle than HT seedlings. An average length of susceptible seedlings was 3 cm, while the average length of HT seedlings was approximately 10 cm (Figure 14, 15, 16, 17). At

10-days, the decay and discoloration symptoms observed in susceptible seedlings was more severe than at 7-days.

The RR-tolerant seedlings displayed normal growth and development in all anatomical structures. These seedlings were considered normal according to the AOSA

Rules for Testing Seed, as well as through a visual comparison to seedlings in the water controls. The RR-tolerant seedlings had no symptoms of toxicity at 7-days (Figure 18).

At 10-days, the RR-tolerant seedlings were evaluated again, and still no toxicity symptoms were observed. Throughout all seed lots and replications there were only a few abnormal seedlings observed. Interestingly, Figure 18: RR tolerant seedling with no symptoms of toxicity next to these abnormal seedlings showed susceptible seedling with toxicity symptoms at 7 days. 32

no phytotoxicity symptoms from the herbicide soak. These abnormal seedlings were

excluded from the calculations.

Calculations for percent Roundup Ready™

RR percentages in a seed lot were calculated by using the number of HT and S

seedlings counted, excluding abnormal and dead seedlings observed. The percentage of

RR sugar beets was calculated using the formula:

% Normal = % Roundup Ready™ % Normal + Susceptible X 100

In all seed lots, the observed counts of normal versus susceptible seedlings were

similar to the expected frequencies. The comparisons for the five frequencies evaluated

are presented in Table 1.

Table 1: Percentage of RR sugar beet seeds in five seed lots tested using the seed soak bioassay method. Each sample was tested using a different ratio of RR seeds to S seeds illustrated below. RR:S RR:S Herbicide # of Abnormal Roundup Seed Lot Expected Normal Susceptible Calculated Concentration Reps & Dead Ready Frequency Frequency 2% 1 4 100:0 391 0 9 = 391/(391+0) 100.00% 2% 2 4 75:25 293 99 8 = 293/(293+99) 74.74% 2% 3 4 50:50 194 200 6 = 194/(194+200) 49.24% 2% 4 4 25:75 96 298 6 = 96/(96+298) 24.37% 2% 5 4 0:100 0 396 4 = 0/(0+396) 0.00%

Pearson’s Chi-square statistic

Hypothesis

Hₒ: The sugar beet seed lot observed RR frequencies are the same as the expected mean

value for each ratio of 75% RR, 50% RR and 25% RR.

Hₐ: The sugar beet seed lot observed RR frequencies are different from the expected

mean value for each ratio of 75% RR, 50% RR and 25% RR.

Table 2: Chi-square for each sugar beet seed lot with the observed value being the normal tolerant seedling result for each seed lot, excluding the 100% RR and 100% S seed lots from Table 1. Expected Expected Expected RR Expected Frequency Observed Chi-square Ratio Frequency Counts

75% RR 293.00 0.75 0.50 291.50 0.0077

50% RR 194.00 0.50 0.33 194.33 0.0006

25% RR 96.00 0.25 0.17 97.17 0.0140

583.00 1.50 1.00 583.00 0.0223

Calculated Chi-square Statistic 0.022298456 degrees of freedom 2 p-value 0.988912694 Calculated in excel with the following formula: =CHISQ.DIST.RT(Calculated Chi-square statistic, degrees of freedom)

The Chi-square statistic value was low at 0.0223 meaning there was high

correlation between the expected and observed data sets. The observed RR frequencies

did not significantly deviate from the expected RR frequency enough to reject our null

hypothesis (p-value is 0.9889). By comparing the p-value of 0.9889 with the significance

level of 0.05 we accepted the null hypothesis because the p-value is greater than the 34

significance level. Furthermore, we accept our null hypothesis that the sugar beet seed lot observed RR frequencies are the same as the expected mean value for each ratio of

75% RR, 50% RR and 25% RR.

Discussion

A seed soak herbicide bioassay is a useful method for determining the percentage of RR seedlings in a seed lot (Gaines, 2020; Goggi & Stahr, 1997). The methodology of submersing the seed in an herbicide solution allows for adequate imbibition of the herbicide for categorizing the seedlings as tolerant, susceptible and abnormal.

Submersing the seeds in an herbicide solution also created less disposal problems for media treated with herbicide. Seeds that were soaked for a period became fragile during the transfer process from the herbicide solution to the planting media (personal observation). Rinsing and blotting the seed dry was an effective method for removing excess herbicide and drying the seed to avoid any physiological damage during planting

(Gaines, 2020). When the moisture content of seed is low and submersed in water or herbicide solution it could create physiological damage to the seed, known as imbibitional injury (LeVan et al., 2008). Damaged seeds germinate poorly due to an increase in seedling abnormalities. Based on a comparison between the germination percentage of the seed lot before and after soaking seed in water (control), there were no signs of imbibitional injury in seedlings or changes in germination percentage in the seed lot. The moisture content of the seed lots used in this experiment was 8% or higher 35

(LeVan et al., 2008), which was expected based on company seed production practices.

Hilleshög Seeds has a target seed moisture content for their seed lots between 8%-10%.

The 2% glyphosate solution proved to be sufficient for differentiating RR and S sugar beet seedlings (Goggi & Stahr, 1997). The herbicide solution had no phytotoxic effects on tolerant seedlings while all susceptible seedlings that germinated showed severe toxicity symptoms. The phytotoxicity symptoms in susceptible seedlings were evident earlier in the seed soak bioassay than in the spray test where the herbicide was applied to the foliage (personal observation). One interesting observation was that RR- tolerant seedlings soaked in herbicide solution appeared to be more vigorous and had healthier roots than the RR-tolerant seedlings in the water control. Gressel and Joel

(2000) describe a process of using glyphosate as a component of priming as well as a seed coating agent to rid the seed lot of contaminating weed seed and provide future immunity to a crop from infection by parasitic weeds in the soil. The priming of sugar beet seed can increase the vigor of a seed lot (Arora, 2017).

The seedlings from the soak bioassay were easy to classify at both, 7 and 10- days. A seed soak bioassay using Roundup™ herbicide can sufficiently be evaluated on day 7 (Gaines, 2020; Goggi & Stahr, 1997; Savoy et al., 2001). Only under circumstances of slow germination would the bioassay need to be extended to 10 days.

The seed soak bioassay significantly reduced the duration of the test when compared to the spray method. When evaluating the bioassay on day 7 it was important to evaluate seedlings in a timely manner and keep the germination trays covered when not evaluating to avoid the PP from drying. As long as lids were closed, there was adequate 36

moisture retained in the PP for up to 10-days. The seed soak bioassay also proved to be a good estimation of seed viability for the seed lot, when compared to the SGT (Savoy et al., 2001). Furthermore, a seed soak in glyphosate could have application in sugar beet breeding programs by screening for glyphosate tolerant plants, evaluating seed lot purity and controlling the purity in backcrossing or self-pollination trials (Shu-feng et al.,

2018).

The PP also was an efficient and effective medium to use for the seed soak bioassay. This medium helped automate the planting and watering of the bioassay, which expedited the process. The efficiency and effectiveness of a seed soak bioassay may encourage the testing of more seeds, which will give a greater confidence level in the RR-purity results (Savoy et al., 2001). The RR and S seed lots used in this experiment had seedlings with both, red and green hypocotyls. This genetic characteristic was advantageous for unbiased evaluation of the bioassay because it made tolerant and susceptible seedlings visually undistinguishable.

CHAPTER Ⅴ

CONCLUSION

The submersion of sugar beet seeds for a 24-hour period in a 2% solution of

Roundup™ Power Max (48.7% a.i.) and subsequent germination in PP proved to be an effective bioassay to determine the frequency of RR seedlings in a seed lot. During the experiment, the susceptible seedlings were clearly distinct from tolerant RR seedlings.

The categories assigned to seedlings (i.e. tolerant vs susceptible) did not change between the evaluation date “day 7” and “day 10” of the experiment. However, on day 10 the susceptible seedlings showed much more necrosis and decay. Furthermore, the readability of the bioassay at day 7 is easy and accurate for an experienced technician.

The seed soak bioassay in sugar beets proved to be accurate, reliable and a cost-effective method for determining the RR-purity percentage in a seed lot. The seed soak bioassay takes less time, labor and resources, which makes this bioassay a good alternative to a spray test. The ability to receive accurate data sooner allows the production, manufacturing and supply chain functions to move forward with purchase orders and conditioning commercial seed with confidence. This experiment demonstrates the seed soak bioassay is a reliable and repeatable method to assess RR frequencies in a sugar beet seed lot.

This experiment only included seed from two known seed lots with high germination. It would be more difficult to determine RR-purity on poor germinating seed lots. A standard operating procedure (SOP) can now be developed by Hilleshög Seed 38

quality assurance lab using the seed soak method and a 2% solution of Roundup™

Power Max. More studies are advised, with the seed soak bioassay, within breeding programs and on all commercial seed lots to prove the methods true usefulness and applicability to the sugar beet seed industry. 39

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