Genetics,PlantBreeding,andSelectionHands-on labs, inc..docx

Genetics, PlantBreeding,
and Selection
Hands-on labs, inc.
Version 42-0063-00-01
Review the safety materials and wear goggles when
working with chemicals. Read the entire exercise
before you begin. Take time to organize the materials
you will need and set aside a safe work space in
which to complete the exercise.
Experiment Summary:
Students will have the opportunity to learn classical
Mendelian Genetics and use a Punnett Square to
predict the outcome of genetic crosses. Students will
perform a monohybrid cross using tobacco mosaic
seeds and study a dihybrid cross using corn. They
will apply chi square analyses to both crosses to
determine the reliability of the data. Students will
design an experiment to produce desired traits in a
hypothetical crop.
© Hands-On Labs, Inc. www.LabPaq.com 248
ExpErimEnt
ObjEctivEs
● To predict the genetic frequency of offspring in a
monohybrid cross

● To predict the outcomes of genetic crosses using Punnett
squares
● To statistically analyze the results of a genetic cross
TimeAllocation: Exercise 1 requires 5–10 days of growing the
tobacco plants prior to performing
the rest of the exercise. Read the exercise and plan accordingly.
Two hours of time is required
after the seeds have been germinated.
www.LabPaq.com 249 ©Hands-On Labs, Inc.
Experiment Genetics, Plant BreedinG, and selection
matErials
MATERiAlS
FRoM:
lABEl oR BoX/
BAg: QTy iTEM DESCRiPTioN:
Student Provides 1 Paper towel
1 Water
1
Warm location for the seeds, either in a warm
window or under an incandescent lamp
1 Roll of aluminum foil
LabPaq Provides 2 Seed, Tobacco, Seeds-Grn/Alb 50

1 Magnifer, dual
1
Pair of tweezers (may be found in dissection
kit)
1 Petri dish, 90 mm
Note: The packaging and/or materials in this LabPaq may differ
slightly from that which is listed
above. For an exact listing of materials, refer to the Contents
List form included in the LabPaq.
DiscussiOn anD rEviEw
Artificial selection is important in plant production. Classical
plant breeding is a process that
uses deliberate interbreeding of plants traits. The plants used
may be closely or distantly related
species, and are interbred to produce new crop varieties. The
goal is to produce more desirable
traits such as flavor, or a resistance to disease or drought.
Artificial selection is also used in breeding
of animals. In some instances, there is no advantage to the plant
or animal; rather selection is
based on human preference. In terms of survivability, it may
actually reduce the success of this
plant’s survival in a natural environment.
Figure 1: These are different cultivars of the same species,
Brassica oleracea, that

have been selected for different traits.
In the real world...
Protoplast fusion is a technique that removes the cell wall from
plants so that cells from two different species can be fused to
create a new species. Embryo rescue is an attempt to save a
weak
or infertile female hybrid by fusing it with more genetically
diverse
male wild plants. The survival rate in embryo rescue varies with
older embryos being more viable. Mutagenesis is the process of
changing the genetic material of a plant through DNA insertion,
chemical or radiation to obtain desired characteristics.
rEviEw Of tErminOlOgy
● Dominance – refers to Mendel’s law that states when two
alleles are present in the
heterozygous condition the dominant allele will mask the
recessive allele.
● Allele – alternate forms of a gene, for example for the trait
freckles the two forms are either
present or absent.
● Dominant – when one allele can dominate or mask the other
allele.
● Recessive - the term given to the allele that is masked. These
are only visible in the phenotype

if they are homozygous recessive.
● Heterozygous – the genotypic condition when both forms of
the alleles (dominant and
recessive) are present in the genotype.
● Homozygous - when the allele pair is the same from the
mother and the father (either both
are dominant or both are recessive).
● Punnett square – tool used to determine the
possible allele combinations that could be
present in a zygote.
● genotype – genes present in an organism.
● Phenotype –physical expression of the alleles or what is
physically seen.
● Monohybrid – a cross between two individuals that differ at
only one trait.
● Dihybrid – a cross between two individuals that differ at two
traits.
Exercise 1: observing a Monohybrid Cross
A monohybrid cross is a simple mixture of traits that uses one
pair of alleles. Dominant traits
will always be expressed when paired with a recessive trait.
Recessive traits are only expressed if

they are homozygous (recessive genes from both the mother and
father) with no dominant allele
present to mask the trait. The dominant trait will be expressed
when dominant and recessive
genes
When a parent with a homozygous dominant gene for height
(TT) is crossed with a parent with
a homozygous recessive gene for height (tt), the offspring will
be 100% heterozygous (dominant
and recessive trait mixture, or Tt). A Punnett square may be
helpful in determining all of the
possible outcomes in offspring when calculating the expected
genotype outcome. A Punnett
square is a simple grid that shows the possible combinations of
the father’s genes on one side
(axis) of the grid and the possible combinations of the mother’s
genes on the other. The grid
can be completed by filling in the combinations of the gametes
possible from the father and the
mother. See examples below.
Figure 2a
Father ( T T ) × Mother ( t t )
Father (♂)
gametes T T
Mother (♀)
t Tt Tt
t Tt Tt
Figure 2b

Father ( T t ) × Mother ( T t )
Father (♂)
gametes T t
Mother (♀)
T TT Tt
t Tt tt
Figure 2: a and b - Parents’ traits shown at top, and their next
generation chart
shown at bottom.
prOcEDurE
Before beginning, set up data tables similar to the Data Tables 1
- 8 in the Lab Report Assistant
section.
Perform these steps at least ten days before seedling data is
required in this laboratory.
1. Cut a piece of paper towel to fit inside a petri dish.
2. Place the paper towel inside the petri dish and moisten with
water. Do not oversaturate
the paper towel (water should not pool in the petri dish); just
moisten the paper towel and

squeeze out excess water.
3. Sprinkle all of the seeds onto the paper towel and then cover
the petri dish with the petri dish
lid.
4. Place seeds near a warm window with indirect light or place
seeds under a warm incandescent
lamp (with the lamp turned on).
a. Lay a piece of aluminum foil over the top of the petri dish if
it is under the light bulb or in
direct sunlight to protect it from the direct light. In order to let
a little light into the petri
dish, just cover the top of the petri dish and allow some light to
filter into the sides of the
petri dish.
5. Observe seeds for germination each day, and moisten the
paper towel as needed. Do not let
the paper towel dry out. Also, only add enough water to make
the paper towel moist, not
oversaturated.
6. There will be two types of F2 seedlings, green and yellow to
very pale green/white which are
albino (chlorotic). If the petri dishes are kept in filtered light
conditions, the green seedlings
should quickly develop chlorophyll (green color) after they
germinate.
7. Count, classify, and record the color of the seedlings in a
data table as they germinate, for
the albino seedlings die quickly. Remove the counted seedlings
with tweezers to minimize
confusion about which seedlings have been counted.

8. Keep the petri dish covered with the plastic lid and limit
exposure to room air as much as
possible to avoid contamination.
Note: It will take 5–10 days for the seeds in this exercise to
germinate.
9. Create a Punnett square (see Data Table 1) that shows the
possible outcomes from a cross
between a heterozygous male and a heterozygous female.
10. Write a hypothesis, using the Punnett square to determine
the approximate number of
seedlings (and percentages) that will have a dominant trait and
that will have a recessive trait
out of the 50 seeds.
11. Once the majority of seeds have germinated, remove the lid
of the petri dish and count the
number of seedlings that are green versus those that are yellow.
12. Record the data into Data Table 2.
13. Compare the expected outcome (Step 7) to actual results
found in Data Table 2 (write this
information into Data Table 3).

Exercise 2: Chi-Square Analysis
Statistical testing may be used to determine the accuracy of test
results. A chi-square test may
determine if the variation between the predicted value and the
actual data is an acceptable level
of variation (less than is expected by chance). Statistical
significance of the chi-square implies that
the difference found between the actual data and the expected
outcome is not due to chance
alone, but instead may be indicative of other variables.
Chi-square analysis uses the following equation, where O = the
observed frequency, E = the
expected count or frequency, Σ = sum, Χ2 = chi-square test:
Chi-square equation:
After obtaining the chi-square value, you will use a chi-square
table to determine significance of
the data. To use the chi-square table, you must determine the
degrees of freedom (df), which
is the number of possible phenotypes [in this exercise, green
and yellow (2)] minus 1. The top
row of the chi-square data table displays the alpha-level, which
is the probability that the two
samples are not different (e.g., 0.05 is a probability of 5%). In
this Exercise, you will conclude that
the samples are different (do not come from the same
population) if the alpha-level is below 0.05
(higher than the chi-square distribution value listed in the
table).

Table 1: Chi square table of probabilities
p value (alpha level)
0.1 0.05 0.01
df chi-square distribution values
1 2.71 3.84 6.63
2 4.61 5.99 9.21
3 6.25 7.81 11.34
4 7.78 9.49 13.28
5 9.24 11.07 15.09
6 10.64 12.59 16.81
7 12.02 14.07 18.48
8 14.36 15.51 20.09
9 14.68 16.92 21.67
10 15.99 18.31 23.21
prOcEDurE
Statistical testing will be used to determine the validity of data
collected in Exercise 1.
1. Record the total number of sprouted seedlings from Exercise
1 in Data Table 4 in the “Total”
row under the columns entitled “Specimens Observed (O)” and
“Specimens Expected (E).”
Note: This must be done after the seedlings are counted. When
using a chi-square test, the total
values (sum of the expected and the sum of the actual value)

must be the same.
2. Determine the “expected values” of the seedlings by
multiplying the total number of sprouted
seedlings by the percentages (ratios) of green and yellow
seedlings that are expected
(determined by the Punnett square in Exercise 1).
3. Record the Expected Values in Column B of Data Table 4.
4. Subtract the observed values in Column A from the expected
values in Column B of Data Table
4, and record these values in Column C.
5. Perform the equation shown in Column D: Use the value
found in Column C, square that
value, and divide it by the Expected Values (E).
6. Add the two values in Column D together (green and yellow)
and record the final sum in the
“Total” row of Column D.
7. To determine the probability that the data values occurred by
chance, use the chi-square
table; see Table 1.
Exercise 3: Dihybrid Crossing with Corn
This is an example of how plant breeding may be analyzed.
In this exercise, you will observe an ear of corn (using photos)

to determine the type of cross
(parents’ genes) responsible for the coloration and texture of the
corn kernels. There are four
grain phenotypes in the ear of corn. The quarter section of corn
from the LabPaq is the result
of a dihybrid cross. Count the number of purple or yellow
kernels and then smooth or wrinkled
kernels. The alleles in this case would be P (dominant purple
color), p (recessive yellow color), S
(smooth shape) and s (wrinkled shape).
Figure 3: Phenotypes in an ear of corn: A) yellow and wrinkled;
B) purple and
smooth; C) purple and wrinkled; D) yellow and smooth.
prOcEDurE
Monohybrid Relationships
1. Count the number of purple and yellow kernels on the ear of
corn between the lines in Figures
4–7.
Figure 4: Phenotypes in an ear of corn: A) yellow and wrinkled;
B) purple and smooth; C)
purple and wrinkled; D) yellow and smooth.
2. Record the data in Data Table 5.
Note: This data represents a cross between a purple kernel corn
plant and a yellow kernel corn
plant.

Figure 5: Ear of Corn, Side A
Figure 6: Ear of Corn, Side C
Figure 7: Ear of Corn, Side D
3. Count the number of smooth and wrinkled seeds on the ear of
corn.
4. Record the data in Data Table 5.
Note: This data represents a cross between a smooth kernel corn
plant and a wrinkled kernel corn
plant.
Dihybrid Cross
5. Construct a Punnett square using Data Table 6.
6. A dihybrid cross involves two alleles, such as PpSs, which
represents a heterozygous dihybrid.
7. The ear of corn is a result of a cross between plants that were
both heterozygous for color and
texture written as PpSs.
8. To create gametes for this type of cross to place on a Punnett
square, use the acronym FOIL

for first (first of the two types), outside (letters on the very
outside), inside (letters on the
inside) and last (last of the two types). An example of alleles
from PpSs crossing with PpSs
would yield:
a. First: PS
b. Outside: Ps
c. Inside: pS
d. Last: ps
9. Place these gametes into the Punnett square in Data Table 6
for both the male and the female.
10. Calculate the phenotypic ratios that are expected to be found
for each type of seed.
a. Purple & smooth _______________
b. Purple & wrinkled ______________
c. Yellow & smooth _______________
d. Yellow & wrinkled ______________
11. Count the number of each on the quarter ear of corn and
record the numbers in Data Table 7.
12. Calculate the expected number based on total number and
assuming the correct ratio. Record
the values in Data Table 8.
13. Calculate the individual chi square values for each row.

14. Add them together to determine the overall chi square value.
15. Determine if the chi square value is a good fit with data.
Note: The degrees of freedom or df is the number of possible
phenotypes minus 1. In this case,
4 – 1 = 3. Using Table 1, find the number in that row that is
closest to the chi square value. Circle
that number.
Genetics, Plant Breeding and Selection
Below is a listing which will help you to prepare for the quiz
and lab on this material.
CONTENT TO KNOW:
I. Terminology used in Genetics
• KARYOTYPES - a method of viewing chromosomes;
o --chromosomes are stained and lined up in pairs;
abnormalities can be seen such as extra or missing
homologues.

• Chromosome Number (humans) - 46 total chromosomes, 23
pairs
homologous chromosomes - alike in size and type of genes
Sex Chromosomes - determine the sex of the fetus
XX - 2 X chromosomes = FEMALE
Xy - one X, 1 y chromosome = MALE
• GENOTYPE = the genetic make-up of the individual
• PHENOTYPE = the physical manifestation of the genotype;
(what the organism shows)
--note: letters are used to represent gene pairs
Dominant allele - A gene which can hide or mask the second
gene
in a pair
Recessive allele - gene which can be masked by the dominant
allele
Ex: W - dominant - widow's peak
w - recessive - straight hairline

3 possible genotypes: WW, Ww, ww
Genotypes:
WW - homozygous dominant
Ww - heterozygous
ww - homozygous recessive
II. Monohybrid Crosses -
Genotypes of the parents:
When you cross a homozygous dominant (WW) with a
homozygous recessive (ww), the results are always:
genotypic ratio - 100% Ww
phenotypic ratio - 100% dominant phenotype
(widow's peak)

Cross two parents that are heterozygous for hairline:
III. DIHYBRID CROSSES -
Dihybrid crosses look at possible combinations of two traits:
Key:
A - purple, dominant
a - white, recessive
B - tall, dominant
b - short, recessive
Cross: AABB x aabb - use the "FOIL" method to determine
gametes: (First, outer,
inner, last)
Results: Parental genotypes are in BOLD FACE along the
top and sides
AB AB AB AB
ab AaBb AaBb AaBb AaBb

Phenotypic ratios:
100% purple, tall
Genotypic Ratios:
100% AaBb
Cross: AaBb x AaBb -
Results: Parental genotypes are in BOLD FACE along the top
and sides
AB Ab aB ab
AB AABB AABb AaBB AaBb
Ab AABb AAbb AaBb Aabb
aB AaBB AaBb aaBB aaBb
ab AaBb Aabb aaBb aabb
Phenotypic ratios:
9 out of 16 are purple, tall
3 out of 16 are purple, short
3 out of 16 are white, tall
1 out of 16 is white, short

Genetics, Plant Breeding, and Selection
Hands-on labs, inc. Version 42-0063-00-01
LAB REPORT
PHOTOS – Include these digital photo with your lab report,
either as a separate attachment to an e-mail or paste into your
document.
1. Photo #1 –Take a photo of the seedlings AFTER they have
sprouted –
close enough to the petri dish to see the little seedlings,
even if they are not clear enough to count in the photo.
Data Table 1: Punnett square
Father ( g g ) × Mother ( g g )
Father (♂)
gametes
g
g

Mother (♀)
Data Table 2: Seedling data
Seedling Color
green
yellow
Total
Number of
Seedlings
Percentage of
Total
Experiment
Genetics, Plant BreedinG, and selection

261
©Hands-On Labs, Inc.
www.LabPaq.com
Questions
A. What are the predicted ratios of the phenotypes in the
plants?
B. What was the total number of seeds that germinated?
C. What are the actual ratios of the phenotypes displayed in
the tobacco plants?
D. Explain why it may be important to collect data from a
larger population.
E. If all yellow seedlings were removed from the population,
would the next generation still have
a chance at displaying the yellow allele? Explain.

F. Estimate the number of generations required before the
yellow alleles were eliminated from the population. Explain.
G. Would a cross between a homozygous and a heterozygous
parent show the same ratios?
Explain.
Exercise 2: Simple Chi-square Analysis
OMIT
Exercise 3: Dihybrid Crossing with Corn
Observations

Data Table 5: Corn kernel data
Number of
Kernels
Kernel Percentage (Divide count by total,
then multiply by 100)
Kernel Coloration
Purple
yellow
Total
Kernel Texture
Smooth
Wrinkled
Total

Data Table 6: Punnet square for dihybrid cross.
Parent gametes ♂
PS
Ps
pS
ps
P a r e n t gametes ♀
PS
Ps
pS

ps
Questions
A. Percent of expected Purple, smooth kernels B. Percent of
expected Purple, wrinkled kernels C. Percent of expected
Yellow, smooth kernels D. Percent of expected Yellow,
wrinkled kernels
Questions
A. What are the probable phenotypes of the corn ear parents
with regard to coloration?
B. What are the probable texture phenotypes of the corn
parents?

Ex
p
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.
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Gen
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tics,
Pla
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, and Selection
Hands
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on
l
a
b
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inc.
V
e
r
sion

42
-
0063
-
00
-
01
LAB REPORT
PHOTOS
–
Include t
hese
digital photo
with your lab report,
either as
a separate
attachment
to an e
-
mail or paste into your document.
1.
Photo #1

–
Take a photo of
the seedlings AFTER they have sprouted
–
E
x
e
r
cise 1:
o
b
se
r
ving
a
Mono
h
ybrid
C
r
oss

D
a
t
a
T
able 1: Punn
e
tt
squa
r
e
F
a
ther
(
g
g )
× Mother
(
g
g )
F
a
ther

(
?
)
g
am
e
t
es
g
g
Mother
(
?
)
D

a
t
a
T
able 2:
Seedling d
a
t
a
Seedling
Color
g
r
een
y
ell
o
w
T
o
t
al
Number of
Seedlings

P
e
r
ce
nt
a
g
e
of
T
o
t
al
Experiment
Genetics, Plant BreedinG, and selection
26
1
©Hands-On Labs, Inc.
www.LabPaq.com
Genetics, Plant Breeding, and Selection

Hands-on labs, inc. Version 42-0063-00-01
LAB REPORT
PHOTOS – Include these digital photo with your lab report,
either as a separate attachment to an e-mail or paste into your
document.
1. Photo #1 –Take a photo of the seedlings AFTER they have
sprouted –
Data Table 1: Punnett square
Father ( g g ) × Mother ( g g )
Father (?)
gametes g g
Mother (?)
Data Table 2: Seedling data
Seedling Color

green yellow Total
Number of
Seedlings
Percentage of
Total

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