Students will map the scientific process of creating a bioengineered (GMO) plant, compare bioengineered soybean seeds to conventional soybean seeds, describe the impact weeds have on plant growth, and understand how a bioengineered seed can help farmers manage weeds. Grades 9-12

agrobacterium tumefaciens: a rod-shaped, gram-negative soil bacterium which can be used to introduce new genes into a plant cell

crossbreeding: selectively breeding two plants or animals of different breeds or cultivars to produce a superior offspring sometimes called a hybrid

farming: the activity or business of growing crops and raising livestock

genetically engineered (GE): an organism or crop whose characteristics have been deliberately modified by manipulating its genetic material

genetically modified (GM): an organism or crop containing genetic material that has been artificially altered so as to produce a desired characteristic

genetically modified organism (GMO): any organism whose genetic material has been altered using genetic engineering techniques

genotype: the genetic makeup of an organism

glyphosate: a synthetic compound (herbicide) commonly used to kill weeds (unwanted plants)

herbicide: a substance that is toxic to plants and is used to kill unwanted vegetation

particle gun bombardment: a method by which foreign substances such as DNA are introduced into living cells and tissues via high-velocity microprojectiles

phenotype: the set of observable characteristics of an organism resulting from the interaction of its genotype with the environment

Roundup herbicide: systemic herbicide and crop desiccant containing Glyphosate used to kill weeds especially annual broadleaf weeds and grasses

tolerant: ability of a plant or animal to endure

transformation: the introduction of exogenous genes into plant cells, tissues, or organs

transgenesis: the process of introducing an exogenous gene, called a transgene, to a living organism

transgenic: containing a gene that has been transferred from one organism to another and acts as a synonym for genetically modified

There are many ways to modify the genes or genome of plants and other living things. These genetic modification tools vary based on which method is needed for acquiring specific traits. This lesson introduces the scientific process used to create a genetically modified organism (GMO) or a bionengineered plant. While GMOs are a topic of socioscientific debate, this lesson focuses solely on the scientific method and biological processes involved in the development of a transgenic plant.

There are many terms and acronyms used to describe genetically modified organisms or biotechnologies applied in plant science. Genetically engineered (GE), genetically modified (GM), bioengineered, GMO, and transgenic are all adjectives used to describe an organism that has a copy of a gene not previously found in the species.

How GMOs Are Created

Every living organism is made from the instructions encoded in strands of DNA contained in its cells. Changes in this genetic code distinguish one species from another (a cow from a corn plant), as well as one trait from another within a species (a cow with a black coat verses a cow with a red coat). Found within the genome of every species is a series of traits and characteristics. Traits manifested through an organism’s phenotype include colors, sizes, and other observable characteristics. Other characteristics such as drought tolerance, resistance to disease, or resistance to chemicals (such as herbicides) are also found within the genotype of an organism. These genetic characteristics are passed from parent to offspring following the basic rules of inheritance originally discovered by Gregor Mendel.

The steps for creating a bioengineered (GMO) plant include the following:⁸

1. The genetic trait of interest is identified in an organism. The gene must first exist in the genome of another organism such as a plant or bacteria.
2. Once the gene responsible for the desired trait is found in a genome, it is isolated.
3. The chosen gene is then inserted into a plasmid using enzymes.
4. Using the process of transformation, the plasmid containing the new gene is inserted into the genome of the plant using protoplasts, plant embryo, or leaf tissue. There are two methods of introducing new genetic material.
   • The particle bombardment method uses a device commonly known as a gene gun. The gene gun shoots metal particles coated with DNA (containing the gene responsible for the trait identified in step one and two above) into the plant tissue.
   • Another method uses a bacteria called Agrobacterium tumefaciens. This bacterium naturally invades plant tissue and alters plants by inserting pieces of its own DNA into the plants genome. This natural process can be used to transfer the transgenic gene into the genome of the desired plant.
5. Once the gene is inserted into the genome of the plant, it is grown. These early stages of plant growth take place in the controlled environment of a greenhouse. As the plant grows, it is carefully observed. In time, scientists will discover if the desired gene was successfully adopted into the genome of the plant.
6. If the newly engineered plant variety contains the desired gene and the adopted trait is successful in its function, the newly engineered seeds are then cross pollinated and the resulting plant offspring are observed for the successful inheritance of the desired trait. The regulatory process will follow to make the seeds commercially available to farmers.

Regulatory Process

After a bioengineered plant is developed, the plant goes through an in-depth series of regulatory tests and trials which are evaluated by the USDA, FDA, and EPA in the United States to determine that the plant and the transgenic gene does not impact the environment in an unintended way or negatively alter the nutritional qualities of the resulting food or animal feed. For example, non-target insects such as pollinators and butterflies are carefully observed to ensure there aren’t any unintended environmental consequences from the newly engineered genome of the plant. Once all regulatory processes are complete, the plant can be approved for commercial use. These regulatory processes are normally carried out by each country that may want to import the seeds to plant or even the foods made from the genetically engineered plant.

The entire process of creating a genetically engineered plant from discovery of the trait to the launch of the seed variety takes several years and can cost over $130 million dollars.¹⁷

Due to the complexity and immense cost of creating genetically engineered seed varieties, transgenesis is not the first tool plant breeders employ to obtain desired plant characteristics.

Weeds and Crop Growth

Weed management is an important factor in agricultural production that impacts crop yield (the amount of a crop that is produced and harvested). Uncontrolled weeds reduce the quantity and quality of a planted crop. Nutrients (found in the soil) and water are necessary, but limited, natural resources for healthy plant growth. Weeds (unwanted plants) compete with planted crops for water and nutrients, thus decreasing the overall harvest and decreasing the efficient use of natural resources.

Farmers can use a variety of weed control methods. There are five general categories of weed control, including preventative, cultural, mechanical, biological, or chemical (with or without biotechnology). Weeds are a constant challenge for every farmer, and herbicides are the primary and most effective tool for farmers to control weeds and maximize crop yields.¹¹ Using herbicides (rather than other weed control methods like hand weeding and tillage) can reduce labor, increase convenience, conserve soil, and make food less expensive to produce.⁹

The Roundup Ready^® soybean was developed in 1990 using biotechnology. In 1996, this biotech crop became commercially available to producers.¹² To develop the Roundup Ready^® soybean, scientists isolated the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) gene from a naturally occurring microbe (Agrobacterium sp. strain CP4) and used particle gun bombardment plant transformation to insert the gene into the genome of soybean (Glycine max). The gene allows the soybean to produce the CP4 EPSPS enzyme that confers tolerance to the herbicide glyphosate, commercially known as Roundup^®. In 2016, over 90 percent of the soybeans grown in the United States were herbicide tolerant.¹³

The Roundup Ready^® soybean was developed to help farmers manage weeds in their fields. When weeds are left to compete with soybeans for the entire growing season, yield losses can exceed 75 percent. Nearly all soybean fields receive some type of herbicide treatment. Monsanto scientists developed the Roundup Ready^® soybean technology as a tool to help farmers control weeds in soybean fields.

Comparing Conventional and GM Soybean

In this lesson students will be comparing the conventional and bioengineered soybeans. Students will examine and compare the seeds as well as the growing plants. There is no visual difference between these two varieties of soybean. The seeds and growing plants will, from a phenotypic standpoint, appear fundamentally the same.

The key difference between the Roundup Ready^® and the conventional seed is that the Roundup Ready^® soybean contains the CP4 EPSPS gene. This gene makes the plant tolerant to glyphosate herbicide. Research conducted by Monsanto and reviewed by global regulatory agencies confirms that the gene results in no other phenotypic changes to the plant.⁷ The presence of the protein CP4 EPSPS can be verified through a lateral flow immunoassay test. This type of test indicates the presence of a protein and is used for many applications, including in-home diagnostic tests like a home pregnancy test. The biotech industry uses lateral flow immunoassay tests in the process of selecting traits in plant breeding, for quality control and purity testing of seed batches, and to support bioengineered labeling. The technique employs the use of protein specific antibodies that bind to a protein (antigen) that is produced by the GMO plant. This protein is produced as a result of the inserted DNA in the transgenic plant, and it is not present in the conventional plant.

Glyphosate (the primary ingredient in Roundup^®) has been used since the 1970s and has been shown to be safe both for the environment as well as in food and feed crops when used according to label directions.¹⁵ Due to Roundup Ready^® soybean’s tolerance to glyphosate (via the CP4 EPSPS enzyme), Roundup^® can be directly applied to soybean fields killing the weeds, but not the soybean. There are many variations of Roundup®. Farmer's use specific formulations of glyphosate to successfully utilize the weed control technology. Formulations of Roundup® available to home owners for lawn and garden use cannot be used successfully due to the addition of other ingredients. Many contain pelargonic acid and diquat dibromide which would kill BOTH the conventional and the Roundup Ready soybean.

1. At the beginning of class, open the What is the Difference? Kahoot survey. Complete the four-question survey with students, allowing them to use personal devices to answer the survey questions.
   • If students do not have access to devices for the Kahoot survey, use the optional attached PowerPoint slides to complete the survey.
2. Following the survey, discuss with students that each difference (illustrated in the 4 pictures) is due to a genetic change. Using our knowledge of science, plant breeders have learned many ways to change and improve the genetic traits of plants. For example, drought tolerant corn (question 1) was developed using cross-breeding. Strawberries (question 2) are larger due to a plant breeding method called polyploidy which increases the number of chromosomes in the plant's genome, and corn plants (question 3) can become genetically resistant to pests like European Corn Borer after acquiring a gene that produces a protein that kills the pest.
3. Explain that these are just 4 examples of desirable traits found in plants. There are many more that range from resistance to disease or herbicides to characteristics of fruit like color or sweetness.
   • If students need additional background about plant breeding, see the lesson plan Methods of Crop Modification.

Teach with Clarity There are many terms and acronyms used to describe genetically modified organisms or biotechnologies applied in plant science. Genetically engineered (GE), genetically modified (GM), bioengineered, GMO, and transgenic are all adjectives used to describe an organism that has a copy of a gene not previously found in the species. This lesson outlines the scientific process of transgenesis where a selected gene is transferred from one organism to another. The BE Disclosure law went into effect in January 2022 and uses "bioengineered" as the term of choice for these products of biotechnology. Throughout the lesson, determine the terminology students are familiar with and provide clarification to prevent misconceptions.

Activity 1: Crops vs. Weeds Simulation

1. Ask students to brainstorm and list ways that farming could be considered a gamble. Students may list factors in agricultural production that are outside of the farmer’s control such as the weather, weed or insect infestation, and market prices. In addition, farmers make dozens of decisions each day that determine the success or failure of their crop. Unlike the weather, these factors are in their control. Examples include choosing which type of seed to plant, seed source, fertilizer type and application, equipment, herbicides, insecticides, and fungicides.
2. Point out that some farmers choose a seed that helps control the weeds that would grow in their field.
3. Explain to students that they are going to do a simulation illustrating why farmers need to control the weed growth in their fields. Each of them will represent a plant. Some students will be a planted crop and others will be weeds. Distinguish between a desired plant and a weed by explaining that weeds are plants that are not desired in a specific growing area. For example, corn growing in a soybean field would be considered a weed.

   Teach with Clarity It's critical for students to understand that weeds in a field of crops are not just undesirable for aesthetic reasons. Weeds reduce overall crop yield and use limited natural resources such as water and nutrients found in the soil.

4. Choose an open area in or around your school to represent a field where your students (simulating plants) are growing. A vacant hallway, outdoor space, or any other large space works well. Explain to students that plants have roots and cannot move to a new location. Throughout the simulation students cannot move their feet, but they can move their arms.
5. Complete Round 1:
   1. Before class, prepare sample green and red cards in a 3:1 ratio to account for the number of students in your class. These cards will help divide your class by assigning 75 percent to be crop plants and 25 percent to be weeds (green=planted crop, red=weed).
   2. Explain that you (the teacher) will play the role of “Mother Nature.” Choose three students from your class to represent the resources of rain, sunlight, and fertilizer. Give each student the poker chips representing their resource (blue=water, red=sunlight, black=fertilizer).
   3. Pass out the green and red cards at random to the remaining members of your class. Line up the students with green cards in rows to simulate a field of planted crop. Students should be far enough apart that they cannot touch when arms are extended. Students with red cards will then “plant” themselves anywhere in the field at random.
   4. Instruct the three students representing resources to sprinkle their “chips” around the feet of the students representing crops and weeds. They should do so after your prompt. For example, “Water, you may make it rain a lot.” [wait a couple of seconds for students to drop blue chips on the floor]. “Okay, the rainstorm is over.” [water stops tossing blue chips.] Continue similar prompts for sunlight and fertilizer. Students (crops and weeds) will scramble to pick up as many poker chips as they can without moving their feet (roots).
      • Note that the three students representing resources may move around as they please during the simulation. For example, one section of the field may receive a lot of sun but no water.
   5. At the end of the round, students who have exactly three of each color may stay standing and go to “harvest.” Students who did not collect three of each chip should sit down to represent the plant dying due to a lack of resources.
      • Note that too much rain, fertilizer, or sun can damage plants.
6. Round 2: Repeat steps outlined above, except begin with 50 percent of the students representing weeds and 50 percent representing planted crops.
7. Round 3: Repeat steps outlined above, except begin with 75 percent weeds and 25 percent planted crops. 
8. Conclude the activity with questions such as:
   • What resources/elements do plants need to survive? (Sunlight, water, and soil nutrients)
   • Why are weeds a problem for farmers? (Water and soil nutrients such as nitrogen, potassium, and phosphorus are in limited supply. If they are consumed to grow weeds instead of food crops, it limits our ability to produce food in an efficient and effective manner.) Display the images of soybeans with and without weed control to illustrate.
   • How can farmers control weed growth in their fields? (Use this question to transition into the next activities.)

Activity 2: How Are Bioengineered Plant Varieties (GMOs) Created?

1. Write the following terms on the board: bioengineered, transgenic, genetically modified (GM), genetically engineered (GE), and genetically modified organism (GMO). These words are often used synonymously.
   • There are many words associated with genetically modified organisms. Clarify the definitions of each term and acronym for better understanding.
2. Ask students to share what these words and acronyms mean to them. Do they know what they mean? Do they have a positive or negative association to the words? Explain that this activity will focus on the science of the GMO and the steps used to create a genetically modified plant.
3. Pass out the How to Create a GMO Interactive Notebook Page. Instruct students to cut out the diagrams and place them in their notebook or on a blank sheet of paper (if you don’t use interactive notebooks) using tape or glue.
4. Beginning with the number one at the top of the diagram, go through each step of the process (left to right, top to bottom). 
5. After you have explained each step of the process, introduce students to an example of a transgenic crop, Roundup Ready^® Soybeans.
6. Give each student one copy of the handout Creating the Roundup Ready^® Soybean. Instruct students to read the handout and create their own diagram representing the steps taken in the creation of the Roundup Ready^® soybean by adding words and illustrations.

Activity 3: How Do You Know it's Bioengineered?

This final activity allows students a hands-on opportunity to see and handle the seeds and/or plants of both conventional soybeans and bioengineered (GMO) soybeans. You may choose to complete just one, or both of the lab activities below. If time is short, choose the GM Soybean Seed Test. If you have additional time and resources to grow the soybeans, continue to the GM Soybean Leaf Test allowing students the unique opportunity to not only compare the seed, but the germination and growth of conventional and GM soybean plants.

GM Soybean Seed Test Lab

*This lab can be completed as a classroom demonstration or by placing students in small groups. For a classroom demonstration, you will need one GM Soybean Seed kit per class. To allow students to perform the test in groups, provide one kit per group.

1. Using the seeds in the GM Soybean Seed Kit, allow students to observe and compare the seeds. Let them know they are the same type of seeds pictured in question #4 of the Kahoot they completed at the beginning of the lesson. Ask students to examine the seeds and look for observable differences in size, shape, and color. Little or no difference will be found in the seeds.
   • Note: As students observe the seeds, be sure the two varieties are kept separate. Place seeds in ziploc bags for observation to help avoid cross contaminating the seeds during observation.
2. Explain to the class that although the two seeds look the same, there is a difference between them, and you are going to do an experiment to discover it.
3. Complete the following lab test for the class to see:
   1. Label the reaction vials for identification of the seed that will be tested by labeling one vial "A" and the other vial "B".
   2. Break the seed by placing one Roundup Ready^® soybean seed between two small weigh boats and tapping it with a hammer. The seed should break into two to three pieces to allow enough surface area to be exposed for extraction. Do not crush the seed. Crushing can cause issues recovering all the pieces for extraction and may cause cross contamination of the testing area. Repeat this step with the conventional soybean, using separate weigh boats to avoid cross-contamination.
   3. Remove the top weigh boats and place the seed pieces into the correct reaction vial. If the seed is stuck to the boat, use tweezers to gently release it. Do not touch the seeds with your hands (clean tweezers with 50-70% ethanol to prevent cross contamination).

      Teach with Clarity It's important to understand that GM seeds are perfectly safe to touch. The only reason you shouldn't in this experiment is to avoid altering the test results by cross contaminating the seed samples or contaminating the experiment with human microbiome.

   4. Use pipette to fill reaction vial with 1% PBS buffer or distilled water (approximately 0.5 mL). Using the pipette as a pestle and the reaction vial as a mortar, stir the seed pieces and distilled water together for 20-30 seconds. Be sure to stir with separate pipettes to avoid cross-contamination.
   5. Let the vial with the seed/distilled water mixture stand for three to five minutes.
   6. Place one QuickStix test strip inside each reaction vial, with the arrow pointing down.
   7. Allow the test to incubate in the reaction vial at room temp for five minutes. While the test incubates, explain how the lateral flow test strip works using the information found in the Background Agricultural Connections section of the lesson. Conclude with students that the test will show a “positive” line if an additional protein is found in one or both of the soybeans. Both test strips should have a "control" line indicating that the test is functioning.
      • Note: You may see positive results in less than five minutes, however the full incubation time will allow for the negative control to fully develop.
   8. Interpreting Results: If the sample contains CP4 EPSPS protein, a second line will develop between the control line and the tape with the arrow on it. If the sample does not contain the CP4 EPSPS protein, a second line will NOT be present on the test strip. 
4. Ask students, “Is there a difference between these two varieties of soybean?” Guide a class discussion for students to conclude that these two varieties of soybean are fundamentally the same. They are both soybeans. However, one variety has a protein that the other does not which gives the plant resistance to the herbicide glyphosate.
5. Ask students, "How did this seed obtain the unique protein?" (transgenesis)

GM Soybean Leaf Test Lab

*This lab can be completed as a classroom demonstration or by placing students in small lab groups. For a classroom demonstration, you will need one GM Soybean Leaf Kit per class. To allow students to perform the test in groups, provide one kit per group.

1. Gather the Roundup Ready^®soybean seeds, conventional soybean seeds, and planting supplies outlined in the Materials section of the lesson. Plant the seeds using the following steps:
   1. Fill tray with soil media, level soil throughout the tray, and saturate with water.
   2. Use a pencil to press a hole into the moist soil 1/2 inch deep.
   3. Drop a soybean seed into each hole and sprinkle soil on top of all seeds. 
      • Note, you can label and separate the Roundup Ready^®  seeds from the conventional soybean seeds, or you could mix the seeds up and plant them randomly without labeling to see if students can distinguish one plant variety from the other.
   4. Place seed tray in a location that receives at least eight hours of bright light per day and maintains warm temperatures of 75-85.° For best results, add water to the bottom tray.
2. Check plants daily for watering and growth. Water trays only if soil is dry. Avoid over watering, but be sure to keep plants from wilting.
3. Students should make regular observations of the germination and growth of the plants. Have students record their observations in their interactive notebook or on a worksheet with column headings such as Date, Temperature, Plant Growth, and Observations.
4. Allow the seedlings to grow for 2-3 weeks or until true leaves appear. (The first two leaf-like structures that emerge from the soil are cotyledons, not true leaves.)
5. Once students have adequately observed the plants and compared them, complete the following lab to test the leaves of the soybean plants for the CP4 EPSPS protein:
   1. Label the microfuge tubes for identification of the plant (leaf) that will be tested by labeling one microfuge "A" and the other microfuge "B."
   2. Obtain 1 leaf from a conventional soybean plant and 1 leaf from a Roundup Ready® soybean plant. Use a true leaf, not a cotyledon (see diagram in Materials section).
   3. Take a leaf punch using the microfuge tube by placing the leaf between the cap and body of the tube. Close the lid of the tube and tear away extra leaf from around the outside of the closed tube. Allow the leaf punch to fall into the tube.
   4. Use the micropestle to push the leaf punch into the bottom of the tube.
      • DO NOT grind the leaf tissue yet! Over extraction can cause chlorophyll to collect in the test band line of the strip and make it difficult to interpret the results.
      • Throughout the lab, use separate tools (micropestle, stir stick, and pipette) for each sample. Cross contamination of the leaf samples will void the test results.
   5. Use pipette to add ~0.5mL of 1% PBS solution or distilled water into each microfuge tube containing leaf tissue.
   6. Macerate the leaf (smash up/grind) using the micro pestle until the leaf is broken into pieces. This doesn’t take much. Once you see some green color in the water the sample has been extracted.
   7. Use the stir stick to mix for 30 seconds. You may also close the lid and shake for 30 seconds.
   8. Place 1 QuickStrip test strip in each reaction vial with the arrow pointing down.
   9. Allow test strip to incubate at room temp for 5 min.
      • Note: You may see positive results earlier than 5 min, however full incubation time will allow for the negative control to fully develop.
   10. Interpreting Results:
       • A single control line should develop on both tests to indicate a successful test. If it does not, dispose of the strip and retest a new sample with a new strip.
       • If the sample contains CP4 EPSPS protein, a second (positive) line will develop.
       • If the sample does not contain the CP4 EPSPS protein, a second line will NOT be present on the test strip.
6. Summarize results with students. Discuss the similarities and differences of the soybean varieties.

*Note: Formulations of Roundup® available to home owners for lawn and garden use typically have additional ingredients that will kill both the conventional and the Roundup Ready® soybean plant. Spraying the plants with Roundup® as a class demonstration is discouraged and will not be successful unless the correct, glyphosate-only spray is used.

Three Dimensional Learning Proficiency Science and Engineering Practices: Asking Questions and Defining Problems Disciplinary Core Ideas: LS1.A: Structure and Function Crosscutting Concepts: Cause and Effect

• Provide students with a scenario. Explain that they operate a farm with several hundred acres of crops. In groups or pairs have students discuss what type of weed control method they will use. Ask students to think, pair, share advantages and disadvantages of using GM seed to manage weeds. 
• Summarize using the following key concepts:
  • A bioengineered (GMO) crop has one or more genes from another organism to create a desired trait. 
  • Transgenesis is the transfer of genes from one organism to another. It is one of several methods of plant breeding and genetic modification.
  • Bioengineered seed varieties have benefits that include tolerance to herbicides to aid in weed control, protection from pests and disease, etc.

1. https://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-us.aspx
2. https://gmoanswers.com/ask/when-did-humans-first-start-genetically-modify-food-and-why
3. http://www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID=23395
4. https://en.wikipedia.org/wiki/History_of_plant_breeding
5. https://www.biofortified.org/2015/07/crop-modification-techniques-infographic/
6. http://www.biologydiscussion.com/plants/protoplasts-plants/protoplast-fusion-meaning-methods-and-its-mechanisms/26481
7. http://www.aphis.usda.gov/brs/aphisdocs/93_25801p.pdf
8. http://sitn.hms.harvard.edu/flash/2015/how-to-make-a-gmo/
9. http://www.agriinfo.in/default.aspx?page=topic&superid=1&topicid=350
10. http://www.acsh.org/news/2016/04/22/crispr-edited-foods-coming-to-a-store-near-you-soon
11. https://extension.psu.edu/introduction-to-weeds-and-herbicides
12. http://www.agbioforum.org/v2n2/v2n2a02-carpenter.htm
13. https://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-us/recent-trends-in-ge-adoption.aspx 
14. https://www.nap.edu/read/10977/chapter/5#43
15. https://www.isaaa.org/resources/publications/briefs/52/download/isaaa-brief-52-2016.pdf
16. https://www.biofortified.org/2014/02/conservation-tillage/
17. http://www.annualreviews.org/doi/abs/10.1146/annurev-arplant-050213-040039

With permission, portions of this lesson were adapted by the National Center for Agricultural Literacy from the original lesson packet, Crop Biotechnology: Growing and Testing Roundup Ready Soybean provided by Monsanto in 2018. The lesson was revised again in 2022.

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