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ABSTRACT
Arabidopsis thaliana, a model system for plant research, serves as the ideal
organism for teaching a variety of basic genetic concepts including inheritance,
genetic variation, segregation, and dominant and recessive traits. Rapid
advances in the field of genetics make understanding foundational concepts,
such as Mendel’s laws, ever more important to today’s biology student.
Coupling these concepts with hands-on learning experiences better engages
students and deepens their understanding of the topic. In our article, we present
a teaching module from the Arabidopsis Biological Resource Center as a tool to
engage students in lab inquiry exploring Mendelian genetics. This includes a
series of protocols and assignments that guide students through growing two
generations of Arabidopsis, making detailed observations of mutant phenotypes,
and determining the inheritance of specific traits, thus providing a hands-on
component to help teach genetics at the middle and high school level.
Key Words: Mendel’slaws;genetics;Arabidopsis; inheritance; phenotype; segregation.
Introduction
Topics related to genetics and genetic modification have been mak-
ing regular appearances in the news for some time now. For today’s
students to develop the scientific literacy necessary to understand
advances in this field, it is important to establish a solid understand-
ing of basic genetic concepts. Presenting challenging content solely
through lectures leaves many students with only a superficial under-
standing of the material (Kontra et al., 2015). By performing hands-
on experiments designed to demonstrate foundational concepts
such as Mendel’s laws, student learning is elevated beyond what
textbooks and lectures alone can accomplish (ACS, 2016; Wyatt &
Ballard, 2007; Zheng, 2006).
Arabidopsis thaliana (Arabidopsis), the first plant to have its
genome completely sequenced, has been transformed from being
just a common weed to serving as a major model system for plant
research worldwide (Somerville & Koornneef, 2002; Koornneef &
Meinke, 2010). Its role expands beyond the laboratory to have
considerable utility in science education. Arabidopsis is a member
of the mustard family (Brassicaceae) and a relative of Wisconsin
Fast Plants, which may be familiar to some science educators.
Arabidopsis, whose common name is mouse-ear cress, provides a
launching point from which students can investigate a wide variety
of scientific concepts, including adaptation to environmental condi-
tions, how plants sense light, and the role of environment and
genetics in growth and development (ABRC, 2016; Provart et al.,
2016). With a short life cycle (6–8 weeks from seed to seed),
self-fertility, and relatively low-maintenance growing requirements,
this plant can be easily incorporated into even the most modestly
equipped science classrooms (Ausubel, 2000; Pang & Meyerowitz,
1987; Zheng, 2006).
The Arabidopsis Biological Resource Center (ABRC) at The Ohio
State University (OSU) is one of two global stock centers providing
seeds, DNA, and other resources to scientists and educators world-
wide. ABRC, which is home to more than 1,000,000 Arabidopsis
stocks, provides samples to approximately 30,000 researchers in
more than 50 countries annually. The Center launched its education
and outreach program in 2011 by releasing 20 teaching modules,
consisting of Arabidopsis seeds and/or DNA resources combined
with lab instructions. The instructional materials have been made
available through ABRC’s education and outreach website (ABRC
Outreach, https://abrcoutreach.osu.edu/), and the seeds and DNA
can be ordered through The Arabidopsis Information Resource
(TAIR, https://www.arabidopsis.org/). The program provides cen-
tralized access to Arabidopsis resources and teaching tools for K-12
and undergraduate education. Seeds are provided free of charge
to K-12 teachers, along with in-depth lesson plans and supplemen-
tal materials that guide educators through the process of incorpo-
rating Arabidopsis into their science curriculum. The activities
presented in this article are based on ABRC’s“Play Mendel”mod-
ule, with additional support for the procedures presented in this
article available on the ABRC Outreach website. Seeds for this les-
son can be ordered from ABRC through TAIR using the module’s
catalog number, CS19985.
The American Biology Teacher, Vol. 80, No 4, pages. 291–300, ISSN 0002-7685, electronic ISSN 1938-4211. ©2018 National Association of Biology Teachers. All rights
reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press’s Reprints and Permissions web page,
www.ucpress.edu/journals.php?p=reprints. DOI: https://doi.org/10.1525/abt.2018.80.4.291.
THE AMERICAN BIOLOGY TEACHER FOLLOWING PHENOTYPES
291
INQUIRY &
INVESTIGATION
Following Phenotypes: An
Exploration of Mendelian Genetics
Using Arabidopsis Plants
•COURTNEY G. PRICE, EMMA M. KNEE,
JULIE A. MILLER, DIANA SHIN, JAMES
MANN, DEBORAH K. CRIST, ERICH
GROTEWOLD, JELENA BRKLJACIC
Lesson Details
This article presents three activities that can be combined or used as
stand-alone units to engage students in the process of growing, breed-
ing, and caring for two generations of Arabidopsis, as well as making
observations, maintaining a detailed lab notebook, collecting data,
and analyzing results. By performing these activities, students gain
insight into concepts such as genotype, phenotype, inheritance, and
segregation of traits, which lie at the core of understanding Mendel’s
laws. The activities are designed for use with middle and high school
classes and are aligned with the Next Generation Science Standards
(NGSS), which are listed within each activity section. This module
provides a structured laboratory experience, and the skills developed
through the procedures and assignments lay the foundation for future
open-inquiry and student-driven experiments using Arabidopsis.
The full lesson (all three activities) spans four months.
Together, these activities guide students through the process of
growing Arabidopsis from the parent (P) generation (Activity 1),
conducting phenotypic analysis of the segregation of a reproduc-
tive trait (Activity 2), and performing genetic crosses to obtain
and analyze the phenotype of the first filial (F1) generation
(Activity 3). Procedures and assignments are listed for each of
the activities (Tables 1, 2, and 4). Students can be engaged in the
entire process for a rich hands-on experience, or portions of the pro-
cedures can be performed by the teachers ahead of time to adapt to
tighter schedules.
Table 1. Schedule of procedures and assignments for Activity 1.
Week Activity Estimated Time Lab Learning Objective
1Procedure 1: Plant P seeds
Assignment 1: Observe growth
1 hour (prep) + 45 min (plant)
20 min 1, 2, 3
2–4Water plants
Assignment 1 continued
20 min twice a week
20 min per week 1, 2, 3
5Water plants
Assignment 1 continued
Assignment 2: ID unique traits
20 min twice a week
20 min
1hour
1, 2, 3
3, 4
Table 2. Schedule of assignments for Activity 2.
Week Activity Estimated Time Lab Learning Objective
6Assignment 1: Analyze inheritance 45 min 1, 2
Table 3. Data table for analysis of the inheritance of select mutations.
Reference
phenotype
Mutant
phenotype Ratio
Group 1
Group 2
Groups 1 & 2
Table 4. Schedule of procedures and assignments for Activity 3.
Week Activity Estimated Time Lab Learning Objective
6Procedure 1: Perform genetic crosses 2 x 45 min 1, 2
7Water plants
Assignment 1: Observe cross outcome
20 min
20 min 1
8–9Do not water plants
Assignment 1 continued
20 min per week 1
10 Procedure 2: Collect F1 seeds 45 min
11–12 No activity
13 Procedure 3: Plant F1 seeds 45 min (prep & plant)
14–15 Water plants 20 min twice a week
16 Water plants
Assignment 2: Compare phenotypes
20 min
45 min 3
THE AMERICAN BIOLOGY TEACHER VOLUME. 80, NO. 4, APRIL 2018
292
Seed Strain Background
•Columbia (Col-1, CS28169)—This is the reference strain of
Arabidopsis. The genome of the closely related Col-0 strain
has been completely sequenced and is used as a basis for com-
parison with other natural strains. Col-1 is a laboratory strain
that has been used to generate many mutants, including the
gl1-1 mutant used in this lesson. Col-1 is used as a reference
strain for the gl1-1 mutant in this lesson.
•gl1-1 (CS28175)—This strain has a mutation in the GLA-
BROUS1 gene, which encodes a protein involved in trichome
(leaf hair) formation. The Col-1 reference strain has tri-
chomes on its stems and leaves. The homozygous gl1-1
mutant is glabrous (hairless), with very few trichomes
present.
•Landsberg erecta (Ler-0, CS20)—This laboratory strain, which
is widely used to generate mutants, carries an X-ray induced
mutation in the ERECTA gene, causing the plant to have a more
upright growth habit. This is the parent strain for the ag-1
mutant used in this lesson. Ler-0 is used as a reference strain
for the ag-1 mutant in this lesson.
•ag-1 (CS25)—This plant has a mutation in the AGAMOUS
gene, which encodes for a protein that controls the produc-
tion of floral organs (sepals, petals, stamens, and carpels) dur-
ing flower development. The term “agamous”means asexual,
referring to the phenotype of the mutant plant, which is ster-
ile. The stamens and carpels in ag-1 mutants have been
replaced by petals and sepals, producing a double flower
(see Figure 2).
Lesson Preparation
Seeds can be ordered from ABRC through TAIR (www.arabidopsis.
org), which requires individuals to register before placing an
order. To streamline the registration process, it is recommended
that first-time users contact ABRC directly to set up an ordering
account with TAIR. To contact ABRC about setting up an
account, e-mail abrcedu@osu.edu with the following information:
full name, email address, school/institution name, job title (ele-
mentary/middle/high school teacher/other), phone number (for
shipping), and shipping address. Seeds should be ordered at least
two weeks before the planned start date of the first activity.
Arabidopsis growsbestat120–150 µmol/m
2
s continuous
light and a temperature of 22–23° C (Rivero et al., 2014).
Detailed protocols for growing and maintaining Arabidopsis
plants can be found on the ABRC website (https://abrc.osu.edu/
seed-handling). Please note that growth rates will vary when
seeds are grown in different conditions. Plants may grow faster
or slower than what is indicated in these protocols if the light
intensity and duration, as well as the temperature, vary from
what is recommended.
Supplemental materials for teachers are provided to facilitate eas-
ier implementation of this unit. Appendix 1 provides definitions of
terms related to plant anatomy and Mendelian genetics. Appendix 2,
designed to serve as a student hand-out, contains simplified labora-
tory protocols for all of the procedures.
Activity 1
Observation of Growth and Development of
Arabidopsis Plants
The first activity in this lesson allows students to observe both
vegetative and reproductive growth and development of Arabi-
dopsis plants over a five-week period. Students will learn about
plant anatomy while making detailed observations throughout
this dynamic period of growth. Instructors can use this activity
to discuss how the development of plant organs, tissues, and cells
contribute to overall anatomy and to identify the different func-
tions each perform in a plant organism. The schedule of proce-
dures and assignments for this activity is listed in Table 1. This
activity is aligned with the following Next Generation Science
Standards (NGSS):
•MS-LS1-4, From molecules to organisms: Structures and
processes
◦Disciplinary Core Idea (DCI) LS1.B, Growth and develop-
ment of organisms
•MS-LS4-4, Biological evolution: Unity & diversity
◦DCI LS4.B, Natural selection
Lab Learning Objectives
1. Make detailed observations of the various growth stages in
mutant and reference plants.
2. Define terms associated with the growing process as well as
with the phenotypes of different Arabidopsis mutants.
3. Compare the phenotypes of mutant and reference strains of
Arabidopsis.
4. Label the anatomy of a plant and identify the role of specific
features in reproduction.
Materials
•4 strains of Arabidopsis seeds (Catalog #: CS28169, CS28175,
CS20, CS25)
•Potting soil
•14-14-14 fertilizer (e.g., Osmocote)
•Teaspoon
•64 plastic pots (Recommended: 1-quart round pots, 4.7″d×
4.75″h)
•8 solid trays (Hummert, Item #11-3050-1)
•8 trays with holes for sub-irrigation (Hummert, Item #11-
3000-1)
•Cheesecloth (Fisher Scientific, Item #06-665-2513) or paper towels
•Weighing boats
•Disposable Pasteur pipettes
•Labeling tape and marker
•Plastic wrap
•Watering can
•Lab notebook
•Growth space with fluorescent lights (http://abrcoutreach.osu.
edu/growing-arabidopsis-classroom)
THE AMERICAN BIOLOGY TEACHER FOLLOWING PHENOTYPES
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Procedure 1: Plant the P Generation Seeds (Week 1)
In this procedure, students will plant two reference strains of
Arabidopsis, a homozygous gl1-1 mutant, and a segregating popu-
lation (heterozygous for gamete formation) of the ag-1 mutant. Divide
the class into two groups, with each group completing the following
planting procedure:
1. Place 1.0 cubic feet of potting soil in a bucket or other large
container and wet thoroughly with water. The moisture con-
tent of the soil should resemble that of a wet sponge. Add
14-14-14 fertilizer to the soil according to the ratio provided
on the product label and blend thoroughly.
2. Line 32 pots with a piece of cheesecloth or paper towel cut to
fit the bottom of the pot such that soil will not escape
through the drainage holes.
3. Place a tray with irrigation holes inside a solid tray. This set
of two trays (one with holes, one without) will be referred to
as a tray throughout the remaining procedures. Label four
trays per group with the group number or name, and the
name of the experiment (glabrous or agamous). Next, label
eight pots with each of the seed stock names (Col-1, gl1-1,
Ler-0, or ag-1).
4. Fill the pots loosely with the prepared soil, taking care not to
limit aeration by compressing the soil.
5. Put a small amount of water in a weighing dish. Select the first
seed stock to be planted and sprinkle a portion of the seeds
into the water. Use a disposable pipette to mix the seeds by
pipetting the water up and down slowly until the seeds are dis-
persed throughout the water. Use the pipette to draw in indi-
vidual seeds and water. Dispense the seeds onto the surface of
the soil, placing nine seeds evenly spaced in each pot. Con-
tinue until you have completed eight pots per seed stock. Do
not cover the seeds with soil.
6. Repeat Step 5 until all four stocks have been planted (Figure 1).
Use a new weighing dish and pipette for each seed stock to
avoid cross-contamination.
7. Place four mutant pots and four of the corresponding refer-
ence strain in each tray. When complete, each group will
have two trays with four pots each of Col-1, and gl1-1, and
two trays with four pots each of Ler-0 and ag-1 (Figure 1).
8. Cover pots tightly with plastic wrap. This will help maintain
humidity until the seeds germinate. If possible, immediately
after covering, place the trays in a dark refrigerator for 2–3
days to promote uniform germination. This process is known
as stratification.
9. After planting (or after stratification), place the trays under
the light source. Once the seeds have germinated and growth
is seen (3–7 days after planting), remove the plastic wrap. Fill
the bottom of the tray with ½ inch of water once or twice a
week. It is important not to overwater the plants or let the
soil dry out completely.
Observation of Genetic Traits
The short life cycle of Arabidopsis provides students with an oppor-
tunity to make detailed observations of the morphological changes
that occur in the plants throughout all stages of growth. To prepare
for the following assignments, have students research the life cycle of
Arabidopsis and become familiar with terms for basic plant structures
and processes (see Appendix 1). There are a number of online
resources where this information can be gathered including the
ABRC Outreach website (http://abrcoutreach.osu.edu/) and the
American Society of Plant Biologists K-12 education page (https://
aspb.org/education-outreach/k12-roots-and-shoots/).
Assignment 1: Observe Growth (Weeks 1–5)
After planting is complete, have students record observations of
the plants in their lab notebooks several times a week for the first
five weeks.
Through this assignment students should:
•Make detailed drawings and notes about each of the four strains
of Arabidopsis.
•Define growth stages of the plants based on the number of
leaves, onset of flowering, silique maturation, and senescence.
•Define terms related to the growing process including stratifi-
cation, germination, bolting, flowering, senescence (see
Appendix 1).
Assignment 2: Identify Unique Traits (Week 5)
Have students compare all four strains of plants to identify the
traits that differentiate each mutant from its corresponding refer-
ence strain. Images in Figure 2 show the distinguishing traits for
each of the four seed strains.
Through this assignment students should:
•Understand the anatomy and function of plant organs.
•Describe the traits using illustrations and notes.
•Record the growth stage when the differences were first noticed.
Figure 1. Illustration of seed spacing and how groups should
organize pots in trays for the experiment. In the Glabrous
experiment, the glabrous (hairless) gl1-1 mutant (containing a
loss-of-function mutation in the GLABROUS or GL1 gene) will
be compared to the Col-1 reference strain (containing a
functional GL1 gene). In the Agamous experiment, the
agamous (asexual) ag-1 mutant (containing a loss-of-function
mutation in the AGAMOUS or AG gene) will be compared to
the Ler-0 reference strain (containing a functional AG gene).
THE AMERICAN BIOLOGY TEACHER VOLUME. 80, NO. 4, APRIL 2018
294
•Compare the two different mutants with respect to growth
stage when each plant’s unique trait was first visible.
Activity 2
Phenotypic Analysis of the Segregation of the
Agamous Trait
An investigation of the inheritance of a mutation in the AGAMOUS
gene can be completed as a stand-alone activity within a single class
session. This activity, which requires only six weeks of grow time,
serves as a demonstration of Mendelian genetics. The schedule of
assignments for Activity 2 is listed in Table 2. This activity is
aligned with the following NGSS:
•MS-LS3-1 & MS-LS3-2, Heredity: Inheritance & variation of
traits
◦DCI LS3.A, Inheritance of traits
◦DCI LS3.B, Variation of traits
•HS-LS3-3, Heredity: Inheritance & variation of traits
◦DCI LS3.B, Variation of traits
Lab Learning Objectives
1. Collect and analyze data to determine the inheritance of a
specific trait in Arabidopsis mutants.
2. Define concepts central to Mendelian genetics (see Appendix 1).
Materials
•2 trays containing 16 pots of 6-weeks old plants (ag-1 and Ler-0,
Figure 1) per group (plants obtained as described in Activity 1)
•Lab notebook
Assignment 1: Analyze Inheritance (Week 6)
In this assignment, students will analyze the inheritance of the ag-1
mutation. Have students determine the number of plants displaying
the reference strain flower phenotype and the mutant flower phe-
notype in the ag-1 pots. Students should arrange their results in a
table (Table 3) and conclude whether this mutation is dominant
or recessive. A dominant mutation requires that only one copy of
the mutant gene be present in order for the mutant phenotype to
be apparent. However, a recessive mutation requires that both cop-
ies of the gene contain the mutation in order for the mutant pheno-
type to be displayed. This activity will demonstrate the importance
of having a statistically significant sample size when analyzing data.
Students will appreciate that the Mendelian ratio of 3:1 may not be
observed with a random sample of small size, but that the ratio
approaches 3:1 with a larger sample. The number of plants of the
segregating ag-1 mutant used in this experiment may not be suffi-
cient for the collected data to accurately reflect the Mendelian ratio.
In this case, teachers are advised either to use the data generated by
previous classes or to reach out online for additional data from
other teachers to aggregate with the data collected by their class.
Teachers can use this experiment as an example to convey the
importance of large sample sizes. Demonstrating the effect of a large
sample size on the outcome of the experiment will also help rein-
force student understanding and appreciation of Gregor Mendel’s
original discovery. Mendel counted and scored phenotypes for
thousands of specimens to conclude the approximate 3:1 ratios.
Through this assignment students should:
•Calculate the ratio of reference to mutant flower phenotypes in
each group.
•Predict what will happen with the ratio if the results from the
two groups are combined.
•Calculate the ratio with the results from both groups combined,
and explain how and why the ratio may have changed.
•Conclude whether the ag-1 mutation is dominant or recessive
(keeping in mind that a segregating population of the ag-1
mutant was planted).
•Use a Punnett square to support the conclusion with evidence
(Figure 3).
Figure 2. Different phenotypes associated with the reference
(Col-1 and Ler-0) and mutant (gl1-1 and ag-1) plants used in this unit.
Figure 3. Punnett square demonstrating the possible
outcomes of a genetic cross in which each parent possesses
one each of the dominant (A) and recessive (a) gene variants
(alleles).
THE AMERICAN BIOLOGY TEACHER FOLLOWING PHENOTYPES
295
At this point in the module, the group is done working with
the Ler-0 and ag-1 plants, and they can be discarded. Once emp-
tied, pots and trays can be disinfected and reused for future proce-
dures in Activity 3. To disinfect pots and trays, dilute ¼ cup of
Lysol per one gallon of warm water. Allow the material to soak
for 10 minutes. Use a sponge or scrub brush to remove any plant
or soil material, then rinse and air dry.
Activity 3
Perform Genetic Crosses to Obtain F1
Heterozygotes for the Glabrous Trait
In this activity students will perform genetic crosses between a gl1-1
mutant and Col-1 reference strain, collect seeds and grow the F1
generation, and analyze the phenotype of the resulting F1 plants.
The schedule of procedures and assignments for this activity is listed
in Table 4. This activity is aligned with the following NGSS:
•MS-LS3-1 & MS-LS3-2, Heredity: Inheritance & variation of
traits
◦DCI LS3.A, Inheritance of traits
◦DCI LS3.B, Variation of traits
•HS-LS3-3, Heredity: Inheritance & variation of traits
◦DCI LS3.B, Variation of traits.
Lab Learning Objectives
1. Generate a segregating population of plants by performing a
genetic cross between mutant and reference plants.
2. Label the anatomy of a plant and identify the role of specific
features in reproduction.
3. Compare the phenotypes of F1 plants to the mutant and ref-
erence strains of Arabidopsis to hypothesize whether the gla-
brous trait is dominant or recessive.
Materials
•2 trays containing 16 pots of 6-week old plants (gl1-1 and Col-1,
Figure 1) per group (plants obtained as described in Activity 1)
•Potting soil
•14-14-14 fertilizer (e.g., Osmocote)
•Teaspoon
•18 plastic pots (Recommended: 1-quart round pots, 4.7″d×
4.75″h)
•3 solid trays (Hummert, Item #11-3050-1)
•3 trays with holes for sub-irrigation (Hummert, Item #11-3000-1)
•Cheesecloth (Fisher Scientific, Item #06-665-2513) or paper
towels
•Weighing boats
•Disposable Pasteur pipettes
•Labeling tape and marker
•Plastic wrap
•Scissors
•Headband magnifier (Lehle Seeds, Item #DA-10)
•Fine-tip tweezers (Lehle Seeds, Item #DV-30)
•Eppendorf tubes (Fisher Scientific, Item #05-408-138)
•Watering can
•Lab notebook
•Growth space with fluorescent lights (http://abrcoutreach.osu.
edu/growing-arabidopsis-classroom)
Procedure 1: Perform Genetic Crosses (Week 6)
Arabidopsis is a self-pollinating plant. Once the flower has opened,
pollination has already occurred. To avoid self-pollination and per-
form a successful cross, students will need to select buds with
barely visible petals to become the female parent plant. Figure 4
demonstrates the various steps outlined in this procedure. This is
a challenging procedure that requires patience. To help prepare
students, have them view the Play Mendel Protocol Video available
on the ABRC Outreach website (http://abrcoutreach.osu.edu/educa-
tional-kits). At this stage of growth, each Arabidopsis plant should
contain multiple flower buds. This will allow students the freedom
to practice preparing a flower for a cross with the understanding
that mistakes will be made, while still providing enough flower
buds for the completion of a successful cross. However, if students
are demonstrating difficulty with this procedure or are unsure of
flower anatomy, teachers may purchase cut flowers with obvious
anatomy (such as lilies) from a grocer or flower shop to use as a
practice specimen.
Figure 4. Steps involved in performing a cross between two
Arabidopsis flowers. Steps A–L are explained in Procedure 1.
THE AMERICAN BIOLOGY TEACHER VOLUME. 80, NO. 4, APRIL 2018
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In this procedure, students will be performing two types of
crosses. First, students will use pollen of a Col-1 reference strain
plant to pollinate a gl1-1 mutant (gl1-1 x Col-1). Then students will
use pollen from a gl1-1 mutant to pollinate a Col-1 reference strain
plant (Col-1 x gl1-1).
1. Have students select a gl1-1 plant and locate an inflorescence
branch containing at least two buds with barely visible petals.
Students should use scissors or tweezers to remove any sili-
ques and open flowers from the branch (Figure 4 A–D).
2. Using a headband magnifier and two pairs of fine-point tweezers,
students should remove the sepals, petals, and stamens from two
buds, being careful not to damage the carpel (Figure 4 E–G). If
the carpel is damaged, remove the flower and proceed to the
next bud.
3. Students should use tweezers to grasp a fully open flower
from the Col-1 reference plant (male parent) at the peduncle
level, squeezing the base to expose the anthers. Students may
then pollinate the gl1-1 female parent by brushing the
anthers of the male parent over the emasculated carpel of
the female flower (Figure 4 H–I). To increase the likelihood
of success, each student should perform multiple crosses.
4. Students should use tape to label the cross and observe the
branch for the formation of siliques (Figure 4 J–K). Let the sili-
ques fully mature (Figure 4 L) before proceeding to Procedure
2. This process normally takes 2–3 weeks after pollination, dur-
ing which time the students will have an opportunity to observe
the development of siliques as part of the next assignment. A
similar procedure should be performed for a reciprocal Col-1
xgl1-1 cross, using Col-1 as a female and gl1-1 as a male parent.
Assignment 1: Observe Cross Outcome (Weeks 7–9)
Have students observe the female parent plants and note the outcome
of the crosses they performed. Approximately 1–2 days after the cross,
the students should be able to tell the difference between a successful
and unsuccessful cross. A successful cross will result in the formation
of a silique (Figure 4 K), an unsuccessful cross will not. Have students
sketch the female parent plant, noting the presence or absence of a
silique in their illustration. Continue to water the plants for approxi-
mately one week after performing the crosses. Stop watering the
plants and let the siliques dry out for at least two weeks, until they
change from green to yellow-brown (Figure 4 L).
Through this assignment students should:
•Illustrate the results of a successful and unsuccessful cross.
•Calculate the percentage of successful crosses for the class.
•Discuss what occurs during the two weeks after pollination.
•Draw the Arabidopsis flower and label the structures listed
below (see Appendix 1). For those structures that have a direct
role in reproduction, have students define that role.
•Stigma, stamen, carpel, inflorescence, petal, silique, sepal,
peduncle, anther, pollen
Procedure 2: Collect F1 Generation Seeds (Week 10)
In this procedure, students will collect the seeds from the successful
crosses and allow them todry out to reduce the internal moisture con-
tent. This drying out process leads to improved seed germination.
1. Use scissors to carefully remove the dry siliques of successful
gl1-1 x Col-1 crosses (F1 generation) and place them in an
Eppendorf tube, one silique per tube. Close tubes and label
with the group number or name, plant number, generation
(F1), and the type of cross.
2. Tap the tube several times to release the seeds from the
siliques.
3. Repeat Steps 1–2 with the siliques of successful Col-1 x gl1-1
crosses.
4. At this point in the module, the group is done working with
the P generation plants and they can be discarded.
5. Allow the F1 seeds to dry for 2 weeks before planting.
Procedure 3: Plant the F1 Generation Seeds (Week 13)
In this procedure students will plant the F1 generation of the gl1-1
x Col-1 and Col-1 x gl1-1 crosses.
1. Following Steps 1–6 outlined in Procedure 1 of Activity 1,
plant the seeds from the gl1-1 x Col-1 and Col-1 x gl1-1
crosses to produce one tray (eight pots) of each type of cross.
Label the pots with your group number or name, plant num-
ber, generation (F1), and the type of cross.
2. In addition, plant one pot each of Col-1 and gl1-1 seeds,
placing 10–20 seeds in each pot. These plants will serve as
controls for phenotypic observations.
3. Follow Steps 8 and 9 in Procedure 1 of Activity 1 for Arabi-
dopsis growth and care.
Assignment 2: Compare Phenotypes (Week 16)
Have students observe and compare the phenotypes of the F1 and
control plants. Through this assignment students should:
•Illustrate and describe the phenotypes of the F1 generation.
•Investigate if any phenotypic differences occur between the F1
plants coming from gl1-1 x Col-1 crosses versus the Col-1 x
gl1-1 crosses. If differences are noted, have students document
those in their illustrations and notes.
•Based on the comparison of the F1 plant phenotype to reference
and mutant plants, have students conclude whether gl1-1 muta-
tion is dominant or recessive.
Conclusions
This unit demonstrates one of many ways that Arabidopsis can be uti-
lized in a science curriculum to reinforce key concepts and engage stu-
dents in hands-on learning. Although originally designed for
instruction at the high school level, this module now aligns withmany
NGSS for other grade levels, which introduce scientific concepts such
as genetic inheritance and variation of traits as early as middle school.
The unit also offers teachers the ability to adjust the depth at which
they cover the material, resulting in a flexible format. Therefore, this
unit can easily be adapted to suit the needs of middle school classes,
as well as advanced placement or other specialized high school biol-
ogy classes, and college-level courses.
Additional experiments that utilize Arabidopsis to demonstrate a
variety of other science concepts can be downloaded from the
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297
ABRC’s Outreach website (https://abrcoutreach.osu.edu/). As stu-
dents become more comfortable with the scientific process, Arabi-
dopsis represents a simple system with which they can design and
conduct their own investigations (Wyatt & Ballard, 2007). Learning
opportunities with Arabidopsis are plentiful, and educators are
encouraged to fully integrate students in the inquiry process using
this model system for plant research.
Acknowledgments
We greatly appreciate the National Science Foundation (NSF) for
their continued support of ABRC, as well as the support received
from the American Society of Plant Biologists (ASPB) to generate
educational resources at ABRC. We recognize and appreciate the
hard work that Marcelo Pomeranz and Nick Holomuzki put into
the initial development of the experiments and original protocols
on which this lesson is based. We are also very grateful to Benson
Lindsey and Nikolas Grotewold for their assistance with the images
contained in this article, and to Chris Bartos for the design of the
ABRC outreach website.
References
American Chemistry Society (ACS). (2016). Importance of Hands-on
Laboratory Science: ACS Position Statement. Available at https://www.
acs.org/content/acs/en/policy/publicpolicies/invest/
computersimulations.html
Arabidopsis Biological Resource Center (ABRC). (2016). Education and
Outreach. Available at https://abrcoutreach.osu.edu/
Ausubel, F. M. (2000). Arabidopsis genome: A milestone in plant biology.
Plant Physiology,124, 1451–1454.
Kontra, C., Lyons, D. J., Fischer, S. M., & Beilock, S. L. (2015). Physical
experience enhances science learning. Psychological Science,26(6),
737–749.
Koornneef, M., & Meinke, D. (2010). The development of Arabidopsis as a
model plant. The Plant Journal,61(6), 909–921.
Pang, P. P., & Meyerowitz, E. M. (1987). Arabidopsis thaliana: A model system
for plant molecular biology. Nature Biotechnology,5(11), 1177–1181.
Provart, N. J., Alonso, J., Assmann, S. M., Bergmann, D., Brady, S. M., Brkljacic,
J., . . . McCourt, P. (2016). 50 years of Arabidopsis research: Highlights
and future directions. New Phytologist,209(3), 921–944.
Rivero, L., Scholl, R., Holomuzki N., Crist, D., Grotewold, E., & Brkljacic, J.
(2014). Handling Arabidopsis plants: Growth, preservation of seeds,
transformation, and genetic crosses. Methods in Molecular Biology,
1062,3–25.
Somerville, C., & Koornneef, M. (2002). A fortunate choice: The history of
Arabidopsis as a model plant. Nature Reviews Genetics,3(11), 883–889.
Wyatt, S., & Ballard, H. E. (2007). Arabidopsis ecotypes: A model for course
projects in organismal plant biology and evolution. American Biology
Teacher,69(8), 477–481.
Zheng, Z. L. (2006). Use of the gl1 mutant and the CA-rop2 transgenic
plants of Arabidopsis thaliana in the biology lecture course. American
Biology Teacher,68(9), 148–153.
COURTNEY PRICE (corresponding author), Education Specialist,
price.1217@osu.edu. EMMA KNEE, Consultant, knee.2@osu.edu. JULIE
MILLER, Program Coordinator, miller.2909@osu.edu. DIANA SHIN, Associate
Curator, shin.260@osu.edu. JAMES MANN, Curator, mann.129@osu.edu.
DEBORAH CRIST, Database and Financial Manager, crist.30@osu.edu. ERICH
GROTEWOLD, Co-Director, grotewold.1@osu.edu. JELENA BRKLJACIC
(corresponding author), Associate Director, brkljacic.1@osu.edu. All are
with The Ohio State University, Arabidopsis Biological Resource Center.
Appendix 1. Key terms and definitions.
General Terms
Reference strain Strain used as the phenotypic benchmark against which mutant phenotypes are compared.
Mutant Strain containing a mutation (a change in the DNA sequence) that is not present in the reference
strain.
Genotype Genetic makeup of an organism that can refer to the specific gene. Examples of different genetic
makeups: TT, Tt, tt represent different variants of that gene (alleles).
Phenotype Physical appearance of an organism for a given trait. Results from the interaction of the genotype
with the environment.
Terms related to Mendelian genetics
Inheritance The process in which genotypes are passed down from one generation to the next
Segregation Separation of pairs of gene variants into reproductive cells
Genetic
variation
Genetic differences found in nature among different individuals of the same species
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Appendix 2. Play Mendel—Handout for students
In this exercise you will plant and observe growth of Arabidopsis thaliana (Arabidopsis), a small plant used as a research model.
The whole genome sequence of this plant is known, enabling scientists to understand the relationship of a gene sequence
(genotype) and the resulting appearance of this plant (phenotype). You will compare the phenotypes of two different mutants
(having a change in the sequence of a mutated gene) to a reference plant with a normal (common, wild type) phenotype. The
first mutant, named gl1-1, has a mutation in the GL1 (GLABROUS1) gene, resulting in the hairless leaf trait. The respective
Col-1 reference plant has leaf hairs. The second mutant, named ag-1, has a mutation in the AG (AGAMOUS) gene, resulting
in a double flower (multiple whorls of sepals and petals) trait. The respective Ler-0 reference plant has a flower with just
one whorl each of sepals and petals. You will have an opportunity to follow these specific traits as they segregate, and make
conclusions about their inheritance based on the analysis of the phenotypes in the progeny resulting from crossing a mutant
with a reference plant. This will help you understand the principles of Mendelian genetics, which are the same for all organisms
with sexual reproduction, including humans.
Activity 1. Observation of Growth and Development of Arabidopsis Plants
Procedure 1: Plant the Parent (P) Generation Seeds
In this procedure, you will work as part of a group. Each group should complete the same procedure.
1. Place potting soil in a bucket and add water and fertilizer. Use gloves when handling fertilizer and fertilized soil.
2. Line the bottom of 32 pots with a piece of cheesecloth or paper towel.
3. Prepare 4 sets of two trays by placing a tray with holes into the one without. Label the four outside trays with your group
number or name. Label two trays “glabrous”and two trays “agamous”. Label eight pots with each of the seed stock names
(Col-1, gl1-1, Ler-0, and ag-1).
4. Fill the pots with soil.
5. Four students should each select one of the seed stocks and fill a weighing dish with water. For each stock, sprinkle
approximately 100 seeds into the water. Use a disposable pipette to mix the seeds by pipetting up and down. Dispense
nine seeds on top of the soil of each pot (see Figure 1). Continue until you have completed eight pots per seed stock.
Do not cover the seeds with soil.
6. Place four gl1-1 mutant pots and four Col-1 pots in each of the two “glabrous”trays. Place four ag-1 mutant pots and four
Ler-0 pots in each of the two “agamous”trays (see Figure 1).
7. Cover trays with plastic wrap and place them in a dark refrigerator for 2–3 days.
Terms related to plant anatomy
Inflorescence A cluster of flowers including the branches of the stem
Peduncle A branch supporting an inflorescence
Stamen Male reproductive organ that includes the anther
Anther The pollen-producing, oval-shaped portion of the stamen
Pollen A carrier of the male reproductive cells. Has an appearance of powder or dust.
Carpel Female reproductive organ that includes the stigma
Stigma The bulb-shaped portion of the carpel, where pollen lands and pollinates the plant
Petal Modified leaves that often function to attract pollinators
Silique Seed pod of the plant
Terms related to processes
Senescence Final developmental stage of the plant; the aging process that leads to death
Stratification Cold treatment of seeds that mimics “winter.”Seeds taken out of cold treatment have higher rates
of uniform germination, a response to “spring.”
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8. After 2–3 days, take the trays out of refrigerator and place them under the light source. Remove the plastic wrap after
seeds have germinated and the seedlings look green. Fill the bottom of the tray with ½ inch of water once or twice a
week. Continue watering the plants while proceeding with the assignments, as instructed by your teacher.
Activity 3: Perform Genetic Crosses to Obtain F1 Heterozygotes for the Glabrous Trait
Procedure 1: Perform Genetic Crosses
After approximately six weeks of growth, the plants should be ready for crosses. Performing crosses on small flowers of a plant
such as Arabidopsis requires patience and practice. Your teacher will show you a video and give you other detailed instructions
to help you master this technique. Don’t be discouraged if you are unsuccessful at the beginning –there are plenty of flowers to
practice on and you will get better!
1. You will start by preparing the gl1-1 flower for crossing. Select an inflorescence branch containing at least two buds with
barely visible petals. Remove open flowers and any siliques from the branch.
2. To expose the carpel (female reproductive organ) of the gl1-1 plant, remove the sepals, petals and stamens from two buds
using a headband magnifier and two pairs of fine point tweezers. If you damage the carpel, remove the flower and pro-
ceed to the next bud.
3. To prepare the pollen of the Col-1 plant, select a fully open flower and use tweezers to squeeze the flower at the base to
expose the anthers (male reproductive organs that carry pollen). Pollinate the prepared carpel of the gl1-1 plant by brush-
ing the anthers of the Col-1 plant over it.
4. Use tape to label the cross and observe the branch for the formation of siliques (seed pods). This is part of the assignment
that you should complete as instructed by your teacher.
5. Perform the same procedure (Procedure 1, steps 1–4) for a reciprocal Col-1 x gl1-1 cross, using Col-1 as a female and
gl1-1 as a male parent. Continue watering the plants for 2–3 weeks until siliques mature.
Procedure 2: Collect F1 Generation Seeds
After 2–3 weeks of silique growth and maturation, the seeds are ready to be collected.
1. Use scissors to remove and carefully place the dry siliques of successful gl1-1 x Col-1 crosses (F1 generation) in an
Eppendorf tube, one silique per tube. Close tubes and label with the group number or name, plant number, generation
(F1) and the type of cross.
2. Tap the tube several times to release the seeds from the siliques.
3. Repeat steps 1–2 with the siliques of successful Col-1 x gl1-1 crosses.
4. Keep the closed tubes in a dry place for two weeks to let the seeds dry out.
Procedure 3: Plant the F1 Generation Seeds
After two weeks of drying, the F1 seeds of both types of crosses are ready for planting.
1. Plant the seeds from the gl1-1 x Col-1 and Col-1 x gl1-1 crosses to produce one tray (eight pots) of each type of cross.
Label the pots with your group number or name, generation (F1) and the type of cross.
2. In addition, plant one pot each of Col-1 and gl1-1 seeds, placing 10-20 seeds in each pot. These plants will serve as
controls for phenotypic observations.
3. Continue watering plants as described in Procedure 1 of Activity 1. After three weeks of growth, your teacher will provide
the instructions related to the assignment at the end of this activity.
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