Abstract

In this single class period case study, students examine the difference between genotype and phenotype by studying the mechanisms by which commercially available petunias have been bred to have different phenotypes (flower colors). Students review the central dogma of molecular biology, connecting the phenotypes of the petunia plants to their genotypes, and the existence of a transgene in plants with unusual (orange) flowers. The case study describes the molecular and biochemical mechanisms by which the secondary metabolites that determine flower color are produced. This case is designed as a “clicker case” utilizing personal response systems or “clickers” to engage students in a large classroom setting. This approach allows for students to collaborate actively, while the instructor is leading and pacing the lecture in a way that allows engagement with large groups. As students work through the case, they work out the processes needed to add transgenes to a plant, as well as how we can detect these modifications decades later. Lastly, instructors can lead a discussion of the scientific and social issues surrounding the creation, use, and regulation of genetically modified organisms.

Primary Image: Orange petunias in Helsinki railway station in 2016. Photo credit: Hany Bashandy and Teemu H. Teeri, CC BY 4.0, via Wikimedia Commons

Citation

Society Learning Goals

Genetics

• Molecular biology of gene function
• How is genetic information expressed so it affects an organism's structure and function?
• Gene Expression and Regulation
• How can gene activity be altered in the absence of DNA changes?
• Genetic variation
• How do different types of mutations affect genes and the corresponding mRNAs and proteins?

Plant Biology

• Plant Genetics and Variation
• Why do individuals of the same species vary in how they look and behave?

Lesson Learning Goals

1. understand the connection between biochemical pathways, genotype, and phenotype.
2. explain the need to regulate the production and sale of genetically modified plants.
3. discuss multiple perspectives regarding genetically modified (GM) plants, their sale, use, and regulation.

Lesson Learning Objectives

1. draw the central dogma of molecular biology, labeling DNA, RNA, protein, DNA polymerase and RNA polymerase.
2. explain how the genotype of an organism may influence biochemical pathways and determine its phenotype.
3. design a process by which a transgene can be inserted into a plant and later identified.
4. evaluate the advantages and disadvantages of regulating genetic modifications in commercial plants.

• explain how the genetic code relates transcription to translation.
• discuss how various factors might influence the relationship between genotype and phenotype (e.g. incomplete penetrance, variable expressivity, and sex-limited phenotype).
• defend how most cells can have the same genetic content and yet have different functions in the body.
• distinguish between loss of function and gain of function mutations and their potential phenotypic consequences.

Introduction

In this single class period case study, students examine the difference between genotype and phenotype by studying the mechanisms by which petunias develop different flower colors. In this case we review central dogma in relation to genotype with the addition of a transgene and the novel phenotype created by this addition. As students work through the case, students should discover the different biochemical pathways involved and the effects of genetic modification (GM) on the phenotypes of petunias. We also discuss the events leading to the mass destruction of orange-flowered GM petunias in 2017 and the public debate surrounding genetic engineering of plants.

Case-based learning uses specific situations, or cases, that are authentic and represent examples of how course content can be applied in the “real world.” In our case we chose the Great Petunia Carnage of 2017, a case of GM of a popular flower that naturally only occurs in pink and purple hues. In the 1980s research was done to add a maize gene to petunias, creating orange-flowered petunias. Decades later, orange-flowered petunias in a train station planter were found to have the same maize transgene. However, since these transgenic plants had not been registered for commercial breeding and sale (1), this discovery led to a worldwide effort to destroy the plants (2). In this case study we explore the mechanisms by which petunia flower color was modified and how these mechanisms represent central dogma of molecular biology (3, 4). We also describe attitudes towards genetically modified organisms and debate multiple sides of the regulation of GM plants. This case is designed as a “clicker case” utilizing personal response systems or “clickers” to engage students in a large classroom setting. This approach allows for students to collaborate actively, while the instructor is leading and pacing the lecture to actively engage students in a way that is not limited by the large classroom setting. Case studies are an accessible teaching method for new faculty, as the structure guides the lesson so even instructors with little experience can adopt case studies successfully and achieve great results with their students (5).

Case-based learning (CBL) is an active and structured approach that is often used to clarify and expand content that students have worked on prior to the introduction of the case. Case-based learning is student-centered and focuses student attention on applying their content knowledge in specific contexts (6). With CBL, students and faculty prepare in advance. They then spend class time in focused discussion of events and perspectives presented in the case-study narrative. Students apply their knowledge in a wider context, developing/creating connections among current subject matter, their prior knowledge, and the social context and application of a given topic (7).

Students learning in a CBL classroom report positive perceptions of their own learning of introductory biology topics in large and small classrooms alike (8). This case study is an ‘interrupted case study’ that features progressive disclosure where information is revealed in small segments, discussed, then evaluated. Students then piece together these bits of information to interpret data and form an informed opinion on the contentious issue of genetic modification in plants. This case study of the Great Petunia Carnage of 2017 also has strong links with topics of ethics and regulatory laws and processes that create an extra layer of authenticity in the classroom.

Intended Audience

We designed this case study for undergraduate students in introductory microbiology, general genetics, or science literacy courses. This case study is also suitable for use in high school biology courses. The case study can be modified, eliminating the more detailed molecular biology concepts when using it in high school or first-year biology courses.

Required Learning Time

This case study is designed for a single classroom session of about 50 minutes.

Prerequisite Student Knowledge

Prior to this case students should be familiar with basic concepts of gene, messenger RNA (mRNA), protein, biochemical pathways, and Polymerase Chain Reaction (PCR).

Prerequisite Teacher Knowledge

While this case study includes adequate background content on the biology of plant cells and the central dogma of molecular biology, teachers may want to review the processes of gene expression and molecular genetics. There are OpenStax textbook sections (such as Chapter 9 of the Concepts of Biology Text) (9) that instructors can use both as a refresher and background for students, depending on the course.

Teachers should also be comfortable using classroom response systems (aka “clickers”) or have a method to capture student data throughout the case progression as information unfolds for the students to evaluate and add to their prior knowledge base.

Scientific Teaching Themes

Active Learning

Teaching case studies inherently promote active learning, with students and faculty discussing particulars of a case, brainstorming ideas, and making connections between concepts and disciplines. In this case, students are asked to answer clicker questions whenever new information is shared by the instructor. The instructor discusses each question set with the group before moving on to the next step in the narrative. If time permits (e.g., in a 75-minute class period), clicker questions can be displayed twice. Students answer individually first, and then they answer the same question again, but in groups of 3 to 4. This leads to students discussing their answers, conferring with peers, and justifying their answers to the group. This interaction is a modified think-pair-share procedure (10) and is similar to the Individual Readiness Assurance Testing (iRAT) and Team Readiness Assurance Testing (tRAT) assessments used in Team-Based Learning (TBL) (11).

Assessment

Students self-evaluate throughout the learning activity as they engage with the content, clicker questions, and think-pair-share activities and group discussion. Additionally, we used a pre- and post-test assessment (Supporting File S1) which consisted of 14 questions that probe student knowledge of the learning objectives. Students took both the pre- and post-test individually, which helps them understand which topics they have mastered, and which topics may need further clarification.

Inclusive Teaching

Incorporation of active learning activities into college science classes increases student achievement overall (12) with disproportionately positive impacts on minoritized groups in STEM (13). Structured active-learning activities, such as case-based learning, are particularly effective for students from minoritized groups in STEM in closing gaps (14).

The collaborative design of this lesson and use of think-pair-share facilitates asset-based approaches, as it encourages cooperation between peers to overcome challenges within the material. Think-pair-share activities are helpful for minoritized students to think and formulate their own answers before they decide to engage with the larger group discussion (15). It is also important to give students enough time to think of an answer to a clicker question, as some students may experience anxiety when they must answer while not having time to really think through their answers (16).

Lesson Plan

Teaching the Case

Pre-Class Preparation

The class period prior to this case, students should take the pre-test to activate their prior knowledge of the topic. If this case is used to introduce the topic of molecular genetics and genetic modifications, the students do not need further preparation, but teachers can assign background reading that describes molecular gene expression (e.g., Chapter 9 of OpenStax Concepts of Biology [9]).

In-Class Presentation

Since this exercise is designed for use in large (100+ student) classrooms, we use an interactive lecture presented as a PowerPoint presentation (Supporting File S2). Slides 2–4 introduce how petunias make colors. In cultivated petunias (Petunia x hybrida), the vast array of different petal colors are the result of differences in flavonoid biosynthesis (17, 18). Flavonoids are plant secondary metabolites, meaning these compounds are not essential for growth or homeostasis, but when naturally occurring often provide a competitive advantage in their environment (19). In addition to attracting pollinators (20), these pigments may protect plants from mutagenesis due to ultraviolet light exposure (21). Flavonoids include flavanols which are colorless compounds and anthocyanins, which are found in a variety of different shades and colors, depending on their chemical structure. Petal and leaf color are influenced by multiple factors, such as anthocyanin concentration within plant cell vacuoles, the pH of vacuoles, and biochemical modifications made by cellular enzymes.

Most of what we know about petunia flower color, shape, and pattern was discovered using a large collection of petunia mutants gathered in the Netherlands in the 1980s (22). The appearance or phenotype of these plants has been described in detail. Different phenotypes are associated with different alleles in key genes influencing the enzymes for pigment production, transcription factors that regulate gene expression, and genes that alter vacuole homeostasis. Most of these genes have been identified by linking mutant genotypes to altered phenotypes. A loss of function mutation in a candidate gene that leads to the loss of a given petal color can place that gene in the pathway the petunia plant uses to produce that color.

The introductory slides are followed by Clicker Questions (CQ) 1 to 4 (slides 5–8) that are designed to test students’ understanding of biochemical processes that determine flower color in petunias. For clicker questions, we displayed each question, allowing students to think about the question and the possible responses before picking an answer. In larger classes, students are often more comfortable discussing course material with 2–3 of their peers than speaking up in a class of a hundred or more. Therefore, students compare notes and impressions they have about the question after answering individually. As the discussion concludes, instructor can shift to a classroom discussion with input from the student groups. Students are generally more comfortable responding to questions with a “My group thinks___” instead of “I think ____.”

Slides 9 and 10 introduce the central dogma of molecular biology, the nature and role of enzymes in petal color production.

Purple-Blue, Dark Red, and Orange Pigment Producing Anthocyanins

There are three types of anthocyanins that determine petal color in petunias and other ornamental flowers. These three are delphinidin-type, cyanidin-type, and pelargonidin-type anthocyanins (23). These different pigments all have a similar core structure that differs in hydroxylation pattern between these three groups. The delphinium-type anthocyanin pigments have three hydroxy groups on their B-ring, which leads to purple fruit (e.g., eggplant, blackberry, grapes) and flowers (e.g., tulips, pansies, petunias). The cyanidin-type anthocyanins have two hydroxy groups which lead to dark red pigmentation in fruits (e.g., pomegranates, red raspberries) and flowers (e.g., red roses). The cyanidin-type are the most common anthocyanins in edible fruits (24, 25). The pelargonidin-type anthocyanins have a single hydroxy group on their B-ring. These anthocyanins are associated with orange and red fruits (e.g., plums, cranberries), flowers (e.g., geraniums, roses) and seeds (e.g., kidney beans). It is important to note that naturally occurring petunias lack pelargonidin-type anthocyanins, meaning that the native cultivars are variations of blue or red pigments, but not orange (23).

After this introduction, CQ#5 (slide 11) asks the students where in the plant to find genes that determine the petal color. It is important to note that this question addresses a common misconception. Students often struggle to understand that a multicellular organism has the same genes in every cell, but different tissues express different genes (26).

Slide 12 specifically addresses this misconception that genes are only found where they are expressed, using the enzyme dihydroflavonol reductase (DFR) as an example. The DFR enzyme is expressed and active in the flowers and fruit of flowering plants, such as petunias.

Slide 13 is CQ#6, which further emphasizes the importance of gene expression, by asking where to find mRNA for DFR. This contrasts with CQ#5, which asked where to find the DNA for the same gene. While the gene is encoded in DNA in chromosomes found in every nucleated cell, the mRNA is only found in tissues that transcribe that gene into mRNA, often as an intermediate for protein production and therefore expression of a version (allele) of a given gene.

Slide 14 reveals details about a research study on flower color that introduced the gene for DFR from maize to petunias that lacked a functional Ht1 gene.

Slide 15 illustrates the process of plant transformation with a gene gun and the p35A1 plasmid that was used to add the maize DFR gene to petunias. The plasmid would usually contain a DNA cassette with the necessary regulatory elements for proper expression within the target tissue and organism, typically a promoter, the gene of interest, a terminator, and a reporter that enables either visual or biochemical selection (e.g., antibiotic or herbicide resistance). Gold particles are coated with the selected plasmid and delivered to leaves using 450 to 900-psi rupture discs, which control the pressure applied to penetrate but not destroy the cells. Subsequently, plants are transferred to soil or regeneration media to recover and grow (27).

Slide 16 is CQ#7, asking students about the nature of plasmids. It is a common misconception that plasmids are structurally different from bacterial chromosomes. Both plasmids and bacterial chromosomes are double stranded DNA, neither are single stranded, and neither are made up of RNA.

Slide 17 is CQ#8, which addresses the requirements for a transgene to be expressed in a new organism. In this case, the transgene was a plant (maize) gene that was then expressed in another plant (petunia). The key takeaway of this question is that plasmids need to include elements that will be recognized by their destination organism, not the organism that provides the gene of interest in a recombinant system. This question is better used in more advanced classes, or if the case study is used as a summary of molecular genetics, not as an introduction to genetics or in a high school biology course.

Slide 18 is exposition, continuing the story by describing the planting of 30,000 transgenic, orange-flowered petunias in Germany and the public’s negative reaction to field release of genetically engineered plants (or merely that scientists were messing about with nature’s creations) (28).

Alternate Methods for Genetic Manipulation in Plants

There have been numerous methods used for genetic modification, from traditional mutagenesis (e.g., x-ray, gamma-ray, fast neutron, ethyl methane sulfonate [EMS]), followed by phenotypic, marker assisted, or genomic selection. These methods do not involve targeted engineering or the use of genes or DNA segments that are not native to the organism that is being modified. However, these mutagenesis efforts leave hundreds to thousands of random mutations in the genome of the treated plant. These can be cleaned up by several generations of backcrossing the mutated plants to their parental lines, but this adds both significant time (years) and monetary barriers to the development of new varieties.

Historically, the most common way of transferring new genetic material was by use of the plant pathogenic bacterium, Agrobacterium tumefaciens. Scientists can use this bacterial pathogen’s molecular machinery and avoid causing disease in target plants by modifying Agrobacterium’s Ti plasmid. Researchers remove the toxin genes from the Ti plasmid, replacing them with a gene of interest, and use the recombinant plasmid as a vector to transfer genes into plant cells (29). However, both Agrobacterium and particle bombardment (gene gun) methods require a vector to integrate the sequence of interest into the host genome. After selection, most of these vectors leave a “foreign DNA footprint.” This has been one of the primary barriers to public acceptance, or regulatory approval of crops that would be used for human consumption.

With the growing use of TALEN or CRISPR-mediated gene editing (30), genes can be targeted with very small changes, even altering a single amino acid in the encoded protein (31). Plants selected for these modifications have the advantage of editing the native genome and negligible “DNA footprints.” This has led to greater public acceptance and regulatory approval, especially in countries that have had strict policies against release of genetically modified organisms (32).

Slide 19 is a short clicker question (CQ#9) asking students to differentiate between genotype and phenotype. While the DFR gene is the genotype of the plant, the orange flowers describe its phenotype.

Slide 20 starts the story of the “great petunia carnage.” A biologist who knew about the 1987 orange petunias sees suspicious looking flowers in a planter at a train station. He takes a stem and a flower to his lab and finds out that this concerns a forbidden flower.

Slide 21 is CQ#10, to answer correctly, students need to consider where the DNA for the maize gene could be found, and how they might determine whether this plant is indeed carrying a transgene and is therefore a genetically modified organism (GMO). This question is also an opportunity for the students and instructor in a more advanced course to discuss some of the more specialized language or jargon associated with molecular biology. For example, blue-white selection is a technique to identify bacteria that have taken up a plasmid with (white) or without (blue) a gene of interest. Given the amount of jargon in this question, instructors may choose omit this question from their presentation.

Slide 22 elaborates on how PCR works and how it can detect minute quantities of DNA in a sample. If the instructors did not use CQ#10, they should make sure to discuss PCR when the class answers the “what would you do if you were Dr. Teeri?” question on slide 20. In slide 23, the story of how the biologist continued to look for GMO petunias continues. Dr. Teeri’s group purchased dozens of different orange petunias from a variety of plant vendors. All of the orange petunias had the maize DFR transgene.

Slide 24 is CQ#11, which asks students to predict the effect of changing a biochemical pathway. The orange petunias made in 1987 had a loss of function in the gene that makes the enzyme that would lead to pink petunias. If this enzyme was functional, the petunias would be pink, and the orange color wouldn’t show in the phenotype because the maize DFR gene doesn’t work very well in petunias (23). Instructors can use this question to stimulate a think-pair-share style discussion, as the actual phenotype is not intuitive, so both orange and pink flowers would be possible, based on what the students know from this case study.

Slide 25 describes the response of the European Union to the identification of these forbidden GMO petunias and CG#12 (slide 26) asks students to reflect on this response and give an opinion. This is an opportunity to discuss the public perception of GMO plants and the role of government agencies in regulating these plants. We started this discussion as a think-pair-share, with students reflecting on the entire case study, forming an opinion, and discussing it in a small group, then discussing as a class.

Slides 27 and 28 show how the debate on forbidden orange petunias continues, as they were finally approved by APHIS and the USDA in 2021, but not without input from the public and scientists.

Details of classroom management can be found in Table 1 and the key for the clicker questions, and their alignment with the learning objectives is found in Supporting File S3.

When this case study was used in a general microbiology course, students took the most time to answer CQ#10 (asking how Dr. Teeri could verify the orange flowered petunia had the maize transgene). When an earlier version of this case study was used in general biology, students struggled with CQ#5 and CQ#6, asking where we could find the DNA and mRNA for a given gene. These questions are an opportunity to reveal and a correct common misconception that only the tissues expressing a gene have that gene.

Table 1. Case study timeline. The case study can be completed in a single 50- or 75-minute class period.

Teaching Discussion

Evidence of Student Learning

We used this case study as an introduction to a unit on molecular genetics and genetic engineering in an intermediate-level microbiology class. Prior to class, students completed a pre-test, and were asked to complete a post-test within 2 days of completing the case study. This test consisted of 14 questions that represented all learning objectives (Supporting File S1). In total 48 students filled out both tests. We tested for normality of distribution and were able to run a paired t test, as the data was normally distributed. The difference between the pre-test and post-test is significant. The effect size shows a moderate effect, which is also reflected in the normalized learning gains (NLG), that are indicative of moderate learning gains (Table 2). A NLG of between 20 and 25 is what may generally be expected of interactive engagement during instruction (33).

We also asked students if they thought they learned more with a case study vs. typical lecture and asked them if they felt more engaged with the course content presented as a case study instead of lecture. Almost 90 percent of the students reported that they learn more when discussing case studies in class than in regular lectures (replying “agree” or “strongly agree” on a Likert scale). Additionally, 80 percent reported they feel more engaged when they discuss case-studies in class. Data was collected after the study was declared exempt by Iowa State University’s Institutional Review Board (IRB - 22-062-00).

Table 2. Summary statistics and normalized learning gains for the Great Petunia Carnage case study.

Suggested Adaptations

This case study can easily be modified for use in non-majors’ microbiology and introductory biology courses by eliminating questions requiring deeper understanding of molecular mechanisms (e.g., CQ#8 and CQ#10). With more detailed information about plant growth, secondary metabolite synthesis and storage, and regulations concerning the construction, release, and commercialization of genetically modified plants, this case study could also be used to introduce a molecular biology unit in a plant biology or horticulture course. This could also be used in a non-majors’ biology course, with less detail about the biochemistry of flower color, or an introductory bioethics course, with more description of the differences in perspective in the United States and Europe in regards to their regulatory standards for genetic modifications in food and non-food plants.

Supporting Materials

• S1. Great Petunia Carnage – Pre- and post-test

• S2. Great Petunia Carnage – Interactive lecture PowerPoint

• S3. Great Petunia Carnage – Clicker questions

Acknowledgments

We would like to thank the Biology of Microorganisms class from Spring 2022 for working through this case study, their lively discussion, and thoughtful suggestions for improvement.

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