Download Lab: Wisconsin Fast Plants Project

Lab: Wisconsin Fast Plants Project
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Purpose
The goals of this lab are to (1) learn how to grow and maintain plants, (2) identify and quantify
phenotypes that are conditioned by a single gene, and (3) understand how Mendelian traits are
inherited.
Introduction
Since the dawn of agriculture, humans have been trying to improve and increase their food
supply. Historically, this was done by selecting the best plants and animals, then breeding
them to get the tastiest, hardiest, most nutritious, or most appealing offspring. But no one really understood how these traits were inherited. Many people believed that traits “blended” in
the offspring, or that only one parent passed a trait on to its offspring.
In the mid-1800s, a monk named Gregor Mendel used the common garden pea to explore his
ideas about inheritance of traits. He articulated two guiding principles of inheritance that remain the bedrock of genetic analysis to this day. The first principle or “law” states that genes
come in two copies (termed alleles) that are segregated during gametogenesis (formation of
sperm and eggs). This is called the Law of Segregation. The second principle or “law” states
that these alleles will assort independently from one another into the gametes only to recombine at fertilization. In other words, if a male carries two different alleles, each sperm he produces with have a 50% chance of receiving one allele and a 50% chance of receiving the other. This is called the Law of Independent Assortment.
Each individual inherits 2 alleles (one from each parent) that together (genotype) determine how
the individual will appear (phenotype). Inherited traits may be described as either dominant or
recessive. The expression of a dominant trait requires just one of the corresponding alleles,
while a recessive trait requires two matching alleles. Another way to think about this is to realize that the recessive trait must be homozygous (two of the same) for the recessive allele, while
the dominant trait may be homozygous or heterozygous (two different alleles). Alleles that confer dominant traits are usually indicated with a capitol letter (G), while alleles that confer recessive traits are indicated with a lower-case letter (g). Therefore, dominant traits would have GG
or Gg genotypes, while recessive traits would be conferred by the genotype gg only.
The goal of this semester-long project is to gather evidence, much like Mendel did, to explain
how inheritance works. Over the course of the project, you will observe 3 different generations
of Wisconsin Fast PlantsTM and collect data about phenotypic traits. You will develop a hypothesis about how you think the phenotypic traits are inherited, perform statistical analyses to
test your hypothesis, and ultimately try to determine the phenotype of the parents of a genetic
cross.
I. Wisconsin Fast PlantsTM Growth and Development
Wisconsin Fast PlantsTM are rapid-cycling Brassica rapa, members of the cabbage and mustard
family. They have a life cycle of 35-40 days (seed-to-seed). They are relatively low maintenance, but will require regular care, including watering (especially on Fridays). As a class we
will decide how to best care for the plants, but ultimately it is your responsibility to keep your
plants alive and healthy over the course of the semester to ensure the success of your project.
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Portions of this lab have been adopted from “Who’s the Father”, Investigating Mendelian Genetics with Wisconsin Fast PlantsTM.
BIO124 Plant Science – Lab Wisconsin Fast Plants Project
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Lab: Wisconsin Fast Plants Project
Anthocyanin is a purple pigment found in many plants. It is best observed in the stems and
hypocotyls, under the cotyledons, and at the leaf tips when plants are 4-7 days old. The anthocyaninless gene (anl) in Wisconsin Fast PlantsTM regulates whether or not anthocyanin will
be expressed. In this experiment, the P1 (mother) and P2 (father) plants are true-breeding,
meaning they are homozygous for their phenotype. The F1 plants are the hybrid offspring that
result from crossing the two parents.
PROCEDURE: (See the Schedule below for corresponding days and dates)
1. Day 1: Plant the F1 and P1 (mother) generations
a. Prepare the water reservoir with algae-control squares. Cover with lid.
b. Saturate the water mat and place on the reservoir lid with ends of the mat extending into the water.
c. Place a wick into each cell of the planting quad so that half of the wick extends
about 2 cm out of the bottom.
d. Moisten the potting soil and fill each quad halfway with soil.
e. Add 2-3 fertilizer pellets to each cell.
f. Fill the quad to the top with more moistened soil.
g. Make a thumb depression in each cell, taking care not to compact the soil too
much.
h. Drop in 2-3 seeds in each depression.
i. Cover seeds with potting mix and water gently with pipette until water drips
from the wick
j. Label each quad with your name and the date and either “F1 generation” or “P1
generation” and place on the water mat.
k. Place the whole reservoir and quads under the lights.
2. Day 2: Score stem color for the F1 and P1 plants and thin plants
a. Score the phenotypes for stem color for the F1 and P2 generations.
b. Discard the P1 plants.
c. Thin the F1 plants to one to two plants per cell. Transplant extra seedlings to
cells with out plants as necessary.
3. Day 3: Intermate F1 plants – this will take several days
a. Make beesticks by placing a drop of glue on the tip of a toothpick. Push the
toothpick into the bottom of the thorax (middle section) of the bee to create a
beestick. Let the beesticks dry completely before use.
b. Pollinate with beesticks by brushing the bee over flowers to pick up and distribute pollen. For 3 consecutive days, use your beesticks to transfer pollen back
and forth among different plants (cross-pollination) because plants do not selfpollinate. Make sure all flowers receive pollen from several different plants.
4. Day 4: Terminate unfertilized flowers
a. Pinch off unopened buds on the last day of pollination and write the date of the
last pollination on the plant labels.
5. Day 5: Stop watering plants
a. 20 days after the last pollination remove plants from the water and allow them to
dry for at least 7 days.
6. Day 6: Harvest seeds from the F1 plants
a. Harvest seeds by gently rolling dry seed pods between your hands over a paper
towel. These are the seeds of the F2 generation.
b. Place the seeds in labeled envelopes and store the seeds in a cool, dry place.
BIO124 Plant Science – Lab Wisconsin Fast Plants Project
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Lab: Wisconsin Fast Plants Project
c. Clean up water reservoirs, platforms, and quads by soaking in a 10% bleach solution. Scrub, rinse, and dry.
7. Day 7: Plant the F2 generation and Hypothesis development
a. Follow the steps for Day 1 to plant the seeds you recently harvested.
b. Develop a hypothesis about how you think the trait controlling anthocyanin expression is inherited. Which trait is dominant (purple/anthocyanin or
green/anthocyaninless)? Which is recessive? Draw out the appropriate Punnett
squares for the F1 generation to demonstrate your hypothesis. Based on your
hypothesis, make predictions about the genotypes and phenotypes of the F2
and P2 (father) plants? Draw out the appropriate Punnett square to demonstrate your expectations for the F2 generation.
8. Day 8: Score stem color for the F2 generation, plant P2 seeds, and perform a chisquare test
a. Follow the steps for Day 2 to plant the seeds for the P2 (parental) generation.
b. Score the phenotype for stem color for the F2 generation.
c. Share your data with the rest of the class.
d. Perform chi-square test with the class data to test your hypothesis. (See section
II below about chi-square tests, we will cover this in lab this day as well.)
e. Interpret the results and draw conclusions about the hypothesis. Was your
hypothesis supported by your data? Speculate on why or why not. Do you accept or reject your hypothesis?
f. If your hypothesis was not supported, modify your hypothesis in view of the
experimental results and repeat the chi-square analysis to further test the modified hypothesis.
9. Day 9: Score stem color for the P2 generation
a. Score the phenotype for stem color for the P2 generation.
b. Does this fit your (revised) hypothesis? Does this fit your expectation from the
chi-square test?
10. WFP Lab Report due November 25 (1 week after analysis of the P2 generation). See
WFP Project and Guidelines and Rubric.
WISCONSIN FAST PLANTS PROJECT SCHEDULE: MONTH August September October DAY 26 2 9 16 23 30 7 14 November December 21 28 4 11 18 25 2 9-­‐13 TOPIC Labor Day – No Lab Day 1: Plant F1 generation Day 2: Score stem color Day 3: Intermate F1 plants Day 4: Terminate unfertilized flowers Fall Break – No Lab Day 5: Stop watering plants Day 6: Harvest seeds Day 7: Plant F2 generation Day 8: Score stem color, Plant P2 seeds Day 9: Score P2 stem color FINAL EXAM WEEK BIO124 Plant Science – Lab Wisconsin Fast Plants Project
ASSIGNMENTS WFP Lab Report Due 3
Lab: Wisconsin Fast Plants Project
II. Monohybrid maize crosses and the chi-square test
Like Mendel’s peas, Zea mays (maize or corn) has been a
popular model system for geneticists studying inheritance.
In the 1930’s Barbara McClintock, with Harriet Creighton,
used maize genetics to demonstrate recombination and later in the 1940’s McClintock discovered transposons, moveable genetic elements. There are several advantages to using maize as a model system, namely a single cross produces many progeny (200+ per ear) making it easy to count
phenotypic ratios and many genes are linked to color markers which can be analyzed in the seed rather than having to
grow up adult plants (Figure 1). Today you will be studying
Figure 1. Maize ears expressing a
a gene, R, that controls seed color and another gene, sugary variety of seed color markers.
(su), that controls starch production in the endosperm of the
seed. The R allele (purple seed) is dominant, while the r allele (colorless or yellow) is recessive.
Similarly Su (starchy or smooth) is dominant, while su (sugary or wrinkly) is recessive. You will
examine these alleles as two independently segregating characters in monohybrid maize
crosses.
A monohybrid cross is a cross between parents who are heterozygous at one locus. The parental cross (P1 x P2) follows: RR x rr. The resulting F1 generation should all be Rr. If you cross
the F1 siblings, Rr x Rr, the result is a monohybrid cross in the F2 generation. Draw out the
Punnett squares for the F1 and F2 generations of this monohybrid cross. Do the same for the
Su trait.
Procedure:
1. With your lab partner, obtain one maize ear segregating purple kernels and sugary kernels.
This ear is the result of a dihybrid cross (i.e. the F2 generation). Observe the variation and relative ratios of seed colors and seed shape.
2. At first glance, are there more purple or yellow seeds, starchy or wrinkly seeds? Develop a
hypothesis about whether you think these two traits are segregating according to Mendel’s
laws. Your hypothesis does not have to be the same for both traits. Your hypothesis should
include the ratios you expect to see per ear for each trait if the gene behaves according to
Mendel’s laws – consider the Punnett square you drew above for a monohybrid cross.
3. Count the number of seeds of each phenotypic class and record your data in a table in
your lab notebook.
4. At this time, you may want to gather data from other student groups. All data is subject to
variability. The larger the data set, the less variability there will be in the average number of
purple kernels per ear for example. You can sum up the total number of seeds for each phenotypic class.
5. Interpret your data. First consider one trait at a time – a monohybrid cross. What is the
ratio of purple kernels to yellow kernels? Does this ratio fit with your hypothesis? Does this
individual trait segregate according to Mendel’s laws? What is the ratio of starchy kernels to
BIO124 Plant Science – Lab Wisconsin Fast Plants Project
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Lab: Wisconsin Fast Plants Project
sugary kernels? Does this ratio fit with your hypothesis? Does this individual trait segregate
according to Mendel’s laws?
7. Perform chi-square tests for each individual trait to statistically determine if the ratios you
observed statistically differ from the ratios you would expect for a monohybrid cross of a trait
that follows Mendel’s two laws. (See the attached explanation of the chi-square test and the
chi-square worksheet.) Are your results statistically different from the expectation?
8. Use what you know about the chi-square test as a statistical way to analyze your genetic
data and apply it to your hypothesis about the Wisconsin Fast PlantsTM.
BIO124 Plant Science – Lab Wisconsin Fast Plants Project
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