Published 2009

Exercise 11 Transmission
(Heredity)
Looking
Genetics
Cell Division (Mitosis)
at Chromosomes

G regor Mendel (18221884) is the founder of the science of heredity, com-

monly known as transmission genetics. His investigations (hobbies) on breeding
Objectives
garden pea (Pisum sativum L.) have been the basis of the 20th century genet-
ics. The scientific and historic views of Mendels work and its implication in
to review
study the
Mendels
stages laws
of
modern genetics can be found in almost all genetic textbooks. In this chapter,
mitosis
of inheritance
in onion root tip
we will not describe Mendelism; rather, we will provide the reader with the
to understand
study the polytene
the terms which are frequently used in transmission genetics and a summary of the
chromosomes
concept of the of
Chi-
the principles of segregation of hereditary traits established by Mendel. To be able
fruit y test
square to understand these principles the reader must have a thorough knowledge of
to learn the genetics of mitosis and meiosis.
Arabidopsis thaliana
Important Terms
to study the inheritance P generation: the parental generation where a specific cross starts
of aleurone color and F1: first filial generation; the progeny of a P P cross
endsoperm shape
F2: second filial generation; the progeny of a F1 F1 cross
in corn
Gametes: germ cells which are haploid; pollen grain and egg in plants and
to study sex- sperm and ovum in animals are germ cells
linked inheritance
True breeding (pure line): an individual self-fertilized several times or crossed
in Drosophila
several times to another individual with an identical genotype (also known as
melanogaster
brother-sister mating) to ensure the traits remain unchanged
to learn setting up Zygote: a diploid cell formed following fertilization
crosses with A. thaliana Allele: alternative form of a gene; alleles could be two or more; if a1 and a2 are
or D. melanogaster the mutations of the same gene, then they are allelic
to learn the technique Wild-type allele: an allele that is found (or seen) in nature; referred to as the nor-
of EMS mutagenesis mal allele; in most cases, wild-type alleles are dominant (see dominant, below)
Mutant allele: a mutated form of the wild-type allele; in most cases, mutant
alleles are recessive (see recessive, below)
Dominant allele (dominant): an allele that masks the expression of another allele
Recessive allele (recessive): an allele that is masked by the expression of
another allele
Incomplete dominance: an allele that is not completely dominant to another
allele, as a result, heterozygous individuals express an intermediate phenotype
Homozygous: an individual carrying the same alleles of a gene either recessive
or dominant; such individual is true bred
Heterozygous: an individual carrying one dominant and one recessive allele of
the same gene; such individual is not a true bred
Genotype: the genetic makeup of an individual; it is made up of thousands of
recessive and dominant alleles
Phenotype: observable characteristics of individuals; that is, how an organism
looks like; it is the combination of expression of dominant and homozygous
recessive alleles
Genome: total amount of genetic material in the chromosomes of an individual

Gregore Koliantz & D.B. Szymanski 62

Types of Crosses
Self-fertilization: a fertilization in which both gametes
come from the same individual
Cross-fertilization: a fertilization in which gametes come
from two different individuals, one serves as male and the
other as female individual
Back cross: a cross between an individual and one of
its parents
Test cross: a cross between an individual and its homozy-
gous recessive parent (or a homozygous recessive individual
carrying the same genotype as the parent)
Reciprocal crosses: crosses in which the male and female
phenotypes (by inference the genotypes) are reversed
Monohybrid cross: a cross in which homozygous parents
differ with regard to the autosomal alleles of a single gene
Dihybrid cross: a cross in which homozygous parents dif-
fer with regard to the autosomal alleles of two genes; and
hence trihybrid cross, tetrahybrid cross, etc.
Sex-linked cross: a cross in which the sex-linked genes of
the parents are sought Fig. 111. A monohybrid cross representing Mendels
Pair mating (in animals): a cross between one male and Principle of Segregation.
one female individual
Mass mating (in animals): a cross between a number of
male and female individuals the parents produce gametes by the process of meiosis (see
Exercise 12), only one copy of the gene will be present in
each gamete: S for the smooth parent and s for the wrinkled.
Mendels Principles It follows that the F1 individuals will be heterozygous for
When two homozygous individuals that differ in only one the trait carrying one copy from each allele (S/s). In the F2
phenotype are crossed together, in the F1 generation, the generation, three types of genotypes are produced, S/S, S/s,
dominant phenotype will be expressed. Upon selfing (that and s/s, with the ratio 1:2:1, respectively (see Fig. 112).
is the F1 F1 cross), approximately 75% of the members This ratio is the result of random fusion of gametes. Since
of the F2 generation will express the dominant phenotype, the S allele is complete dominant to the s, the S/S and S/s
and 25% will show the recessive phenotype. The result individuals will be phenotypically identical. As a result, the
will be the same if the reciprocal cross is involved. Figure genotypic ratio 1:2:1 will be shifted to the phenotypic ratio
111 illustrates a cross in the garden pea showing smooth 3:1. If the S allele were incomplete dominant to the s, then
or wrinkled seeds. From this cross the following rules can the genotypic and phenotypic ratios would have been the
be extracted: same: 1:2:1.
1. The outcome of reciprocal crosses is the same. This sim-
ilarity is referred to as the Principle of Uniformity.
2. In the F1 generation, the dominant phenotype is ex-
pressed.
3. In the F2 generation, the parental phenotypes reappear
with the ratio of 3:1, where the represents the domi-
nant phenotype (the smoothness of the seeds), and the
shows the recessive phenotype (the wrinkledness of
the seeds). This phenotypic segregation represents the
Principle of Segregation of the alleles.
An alternative way to show the cross displayed in Fig.
111 is to use letters to represent the alleles. The letter S can
be assigned to smoothness and the letter s to wrinkledness.
Note that the dominant allele is written with capital letters
and recessive allele with lowercase letters (Fig. 112).
Since the parents (P) are homozygous for the trait, they
are represented with two copies of the same allele: S/S for
the dominant allele and s/s for the recessive allele. When Fig. 112. The same cross as in Fig. 111. Here, letters
demonstrate the Principle of Segregation.

63 Genetics: A Laboratory Manual

 Consider the following example. Suppose in
a population of 218 plants, you counted 162 indi-
viduals with red and 56 with white flowers. These
plants had been generated by a cross between two
heterozygous parents. Your expectation is that the
progeny to appear in a 3 red and 1 white ratio. On
this basis, the expected number of plants with red
flowers would be 218 3/4 = 163.5 and that of white
flowers 218 1/4 = 54.5.
Given the Chi-square formula
2 = (O E)2/E
where is the sum of all values, O is the observed
(actual) numbers, and E is the expected numbers (to
be calculated); the Chi-square can be calculated as
shown in Table 111a. However, in this example, if
your expected ratio is 1 red and 1 white, then the
expected number of red and white flowers would be
the same, that is 218 1/2 = 109. Under this assump-
Fig. 113. Independent assortment of yellow-starchy (C/C;Su/Su) tion, the Chi-square will be calculated according to
and purple-sweet (c/c;su/su) kernels in corn.
Table 111b. Note that in Chi-square tests data
are reported as whole numbers.
Mendel extended his investigations beyond monohybrid To find the level of significance of the 2 values
crosses. He set up crosses between the pea plants that differ shown in Tables 111a and 111b, the Chi-square table is
in two phenotypes; for example, plants with smooth and used (Table 112).
yellow seeds vs. plants with wrinkled and green seeds,
typically a dihybrid cross. Consequently, he proposed the How to Use the Chi-Square Table
Principle of Independent Assortment which states that 1. Determine the degrees of freedom. In general, the de-
the alleles of different genes are combined independently grees of freedom indicate the number of phenotypic
of each other. Today, we know that independent assortment groups that are independent of each other. In Tables
is possible only if the genes involved are located on different 111a and 111b, there are two phenotypic groups:
chromosomes (see Fig. 113) and that these genes segregate plants with red flowers and plants with white flowers.
independently in the production of gametes. However, some Since the total number of plants is 218, of which 162
of the traits Mendel studied were located on the same chro- have red flowers, the number of plants showing white
mosome (i.e., linked), but they segregated independently
because of the occurrence of crossing over (see Exercise 12)
between them. Mendel was unaware of these events. Table 111a. The Chi-square test for a 3:1 segregation
of the trait.
To illustrate the Principle of Independent Assortment,
a cross is set up between homozygous yellow-starchy Red White Total
Observed (O) 162 56 218
(C/C;Su/Su) and purple-sweet (c/c;su/su) corn. All the
Expected ratio 3/4 1/4
individuals of the F1 generation will follow the rule of Expected (E) 163.5 54.5 218
Uniformity and the dominant phenotype (yellow-starchy) OE 1.5 1.5 0
will be expressed (Fig. 113). The F1 individuals, which (O E)2 2.25 2.25 4.50
are heterozygous for both alleles (C/c; Su/su), will develop (O E)2/E 0.013 0.041 0.054
four types of gametes because each C or c allele will segre- 2 = 0.054
gate independently of Su or su allele. Consequently, upon df = 1
random fusion of these gametes, 16 (4 4) genotypic com-
binations will occur in the F2 generation (see Fig. 113). Table 111b. The Chi-square test for a 1:1 segregation of
These 16 combinations will be grouped in four phenotypic the trait.
classes because of the dominance, giving rise to a 9:3:3:1 Red White Total
phenotypic ratio. Observed (O) 162 56 218
Expected ratio 1/2 1/2
Expected (E) 109 109 218
Statistical Treatment of Data: OE 53 53 218
The Chi-Square Test (O E)2 2809 2809 5618
The Chi-square (2) test is often used in genetics to (O E)2/E 25.770 25.770 51.54
test the significance of deviation between observed and 2 = 51.54
expected numbers. df = 1

Gregore Koliantz & D.B. Szymanski 64

Table 112. Chi-square values. The arrow indicates the split point for accepting or as those of crop plants, but
rejecting a hypothesis. Critical values are shown in bold font. it has a shorter generation
time. These features make
Degrees Probabilities Arabidopsis an amenable
of freedom 0.95 0.80 0.50 0.10 0.05 0.01 0.001 organism for plant genetics.
acceptreject However, Arabidopsis has
1 0.00 0.06 0.46 2.71 3.84 6.64 10.83 no agronomic significance.
2 0.10 0.45 1.39 4.61 5.99 9.21 13.82 Arabidopsis is a self-fertile
3 0.35 1.01 2.37 6.25 7.82 11.35 16.27 plant; a single plant can
4 0.71 1.65 3.36 7.78 9.49 13.28 18.47 produce more than 100
5 1.15 2.34 4.35 9.24 11.07 15.09 20.52
6 1.64 3.10 5.35 10.65 12.59 16.81 22.46 seed pods or siliques. Each
7 2.20 3.82 6.35 12.02 14.07 18.48 24.32 healthy silique contains
8 2.73 4.60 7.34 13.36 15.51 20.09 26.13 about 40 to 50 seeds. From
9 3.32 5.40 8.34 14.68 16.92 21.67 27.88 a healthy wild-type plant,
10 4.00 6.20 9.34 15.99 18.31 23.21 29.59 thousands of seeds can be
Originally from R.A. Fisher and F. Yates (1943). obtained. The entire life
cycle of Arabidopsis (from
seed imbibition to seed
flowers has to be 56 (i.e., 218 162). Therefore, the harvest) is completed in
latter is not an independent group. In other words, if about 6 to 7 weeks. Table 113 shows the growth stages of
there are two phenotypic groups, only one group is Arabidopsis from leaf development through seed maturation.
independent, and there is only one degree of freedom. The data have been collected from the Columbia wild-type
In general, the degrees of freedom are equal to the strain grown under a 16-h light, 8-h dark cycle.
number of phenotypic groups minus one. Arabidopsis grows in the wild throughout temperate
2. In Table 112, use the probability of 0.05 as a split regions of Europe, Asia, and North Africa on sandy or
point for accepting or rejecting a hypothesis and note gravely soil. In the greenhouse, Arabidopsis grows very
the critical values shown in that column. well on soilcompost mixes at temperatures in the range of
3. Since in this example, there are two phenotypic groups, 22 to 26C and in a relative humidity of around 50%. Lower
in Table 112 look at the first row where there is one temperatures slow growth. Arabidopsis requires a 12 h or
degree of freedom and find the corresponding critical greater photoperiod to flower. We raise Arabidopsis under
value (in this case it is 3.84). The calculated 2 value continuous light at 25C in a relative humidity of 50%.
for the 3:1 segregation in Table 111a is 0.054, which Plant fertilizers, dissolved in water, can be used to feed
is less than 3.84; therefore, you fail to reject the hy- Arabidopsis; however, excess of watering may reduce plant
pothesis of the 3:1 segregation. On the contrary, the hight, can cause early flowering, and reduced productivity.
calculated 2 value for the 1:1 segregation in Table
111b is far greater than 3.84; therefore, you are in-
clined to reject the hypothesis of 1:1 segregation.
Fig.114. (A) A mature
plant of Arabidopsis
thaliana grown in the
Exercises with Arabidopsis thaliana greenhouse. (B) Floral
The genus Arabidopsis is a member of the Brassicaceae diagram of A. thaliana.
(mustard or crucifer) family and has nine species and eight
subspecies. The most studied species is Arabidopsis thali-
ana, or thale cress (also known as wall cress or mouse-ear
cress). Arabidopsis thaliana (hereafter called Arabidopsis
unless otherwise specified) is a dicotyledonous angiosperm;
that is, the mature embryo gives rise to two cotyledons
(dicotyledon or dicot) and the seeds are enclosed within an
ovary (angiosperm).
Vegetative growth of Arabidopsis is marked by rise of the
radicle followed by the formation of hypocotyls and coty-
ledons from seed coat. After cotyledons are fully opened,
rosette leaves are formed. Emergence of the first flower bud
indicates the beginning of the reproductive growth of the
plant. Flowers are white in color, about 3 mm long, and pro-
duce four sepals, four petals, six stamens, and a single ovary
consisting of two fused carpels (Fig. 114). Developmental
and physiological aspects of Arabidopsis are almost the same

65 Genetics: A Laboratory Manual

 The most widely used Table 113. Arabidopsis growth stages for the soil-based phenotypic analysis plat-
Arabidopsis ecotypes (i.e., form. Reprinted from The Plant Cell 13:14991510 by permission of the American
strains isolated from differ- Society of Plant Biologists.
ent geographical regions) are
Columbia (Col) and Landsberg Col-0 data
erecta (Ler). Both ecotypes were Stage Description Days SD CV
derived from the same popula- Principal growth stage 1 Leaf development
tion originally named Landsberg. 1.02 2 rosette leaves > 1 mm in length 12.5 1.3 10.7
Columbia was derived by 1.03 3 rosette leaves > 1 mm in length 15.9 1.5 9.5
self-pollination from a single 1.04 4 rosette leaves > 1 mm in length 16.5 1.6 9.8
plant with high reproductivity. 1.05 5 rosette leaves > 1 mm in length 17.7 1.8 10.2
1.06 6 rosette leaves > 1 mm in length 18.4 1.8 9.8
Landsberg erecta was selected
1.07 7 rosette leaves > 1 mm in length 19.4 2.2 11.1
for its short and erect stature 1.08 8 rosette leaves > 1 mm in length 20.0 2.2 11.2
following gamma irradiation. 1.09 9 rosette leaves > 1 mm in length 21.1 2.3 10.8
A number of other ecotypes are 1.10 10 rosette leaves > 1 mm in length 21.6 2.3 10.9
also available which are adapted 1.11 11 rosette leaves > 1 mm in length 22.2 2.5 11.2
to specific ecosystems around 1.12 12 rosette leaves > 1 mm in length 23.3 2.6 11.3
1.13 13 rosette leaves > 1 mm in length 24.8 3.2 12.8
the world. These ecotypes 1.14 14 rosette leaves > 1 mm in length 25.5 2.6 10.2
include, but are not limited to, Principal growth stage 3 Rosette growth
Wassilewskija (Ws), Eastland, 3.20 Rosette is 20% of final size 18.9 3.0 16.0
Dijon G, and C24. 3.50 Rosette is 50% of final size 24.0 4.1 17.0
3.70 Rosette is 70% of final size 27.4 4.1 15.0
History 3.90 Rosette growth complete 29.3 3.5 12.0
Arabidopsis thaliana was first Principal growth stage 5 Inflorescence emergence
identified by Johaness Thal in the 5.10 First flower buds visible 26.0 3.5 13.3
Principal growth stage 6 Flower production
1570s. In 1873, a mutant (believed 6.00 First flower open 31.8 3.6 13.3
to be agamous: whorls of the sta- 6.10 10% of flowers to be produced have opened 35.9 4.9 13.6
mens and gynoecium replaced by 6.30 30% of flowers to be produced have opened 40.1 4.9 12.3
extra whorls of petals and sepals) 6.50 50% of flowers to be produced have opened 43.5 4.9 11.2
Arabidopsis plant was found in 6.90 Flowering complete 49.4 5.8 11.7
Europe. In 1943, Arabidopsis was Principal growth stage 8 Silique ripening
8.00 First silique shattered 48.0 4.5 9.3
considered as a model system Principal growth stage 9 Senescence
in genetics and the first major 9.70 Senescence complete; ready for seed harvest ND ND ND
review article was published by
G. Redei in 1970. Thirteen years Average day from date of sowing, including a 3-d stratification at 4C to synchronize germination.
CV, coefficient of variation, calculated as (SD/day) 100.
later, the first detailed genetic
ND, not determined.
map of Arabidopsis was pub-
lished. It was not until 1990 that a high number of genes: 30,918 compared with 30,887 in
the Arabidopsis Genome Project was initiated which led to the Drosophila melanogaster, 7547 in Saccharomyces cere-
completion of the physical map of all five chromosomes in visiae (yeast), 23,399 in Caenorhabditis elegans (worm),
1997. During the years 1999 and 2000, the entire Arabidopsis 12,792 (predicted) in Danio rerio (zebrafish), and 31,896
genome was sequenced and the existence of some 25,500 in humans. This high number is due to the fact that about
genes was predicted. However, to date 27,029 protein coding 58 to 60% of the genome is duplicated, likely because of
genes and 3889 transposable elements have been identified, independent duplication events that took place during the
raising the total number of genes to 30,918. evolution of this species.
Model Organism
Arabidopsis thaliana has a number of advantages that make Mutants
it an amenable organism for genetic research: In Arabidopsis, thousands of mutations have been identi-
it is a relatively small plant fied which alter developmental progression or result in
it reproduces rapidly morphological and physiological changes.
it is convenient to grow in the laboratory
it has a relatively small genome Mutations occur spontaneously; however, they can be
its genome has been sequenced induced by chemicals such as EMS (see Protocol 113), irra-
it can be easily transformed by Agrobacterium tumefaciens, diation with gamma or X rays, or T-DNA insertion. To ensure
creating transgenic plants (see Protocol 101) that the mutant phenotype is indeed due to a single mutation,
The Genome the mutant plant must be backcrossed at least four times to a
Arabidopsis thaliana is a diploid plant with a diploid number wild-type strain and the progeny selected for the mutant phe-
of 10 chromosomes (2n = 10). Arabidopsis has surprisingly notype. Table 114 lists a variety of Arabidopsis mutations.

Gregore Koliantz & D.B. Szymanski 66

Table 114. Representatives of Arabidopsis thaliana mutations.
Gene Map
symbol Name Phenotype Chromosome position
cM
cer eceriferum Very bright green stem and silique, reduced plant height 1 70.1
an-1 angustifolia Narrow leaves, trichomes unbranched 1 55.2
er erecta Upright stem, compact inflorescence, short petioles 2 43.5
py-201 pyrimidine requiring Requires thiamine, leaves of unsupplemented plants white or pale 2 53.0
gl1 glabra No trichomes on stem or leaves 3 46.2
spy-5 spindly Early flowering, usually light green, somewhat curled leaves, bent siliques 3 12.0
bp-1 brevipedicellus Short pedicels, siliques bent downwards, plant height reduced 4 15.0
im immutans Variegated phenotype 4 48.0
pi pistillate Lacks petals and anthers, maintained as heterozygote 5 21.0
ch chlorine Yellowgreen plant 5 43.0

National and International Resources Materials

The Arabidopsis Information Resource (TAIR), provides Hand magnifying glass
genomic and literature data, and can be found at http://www. Dissecting microscope
arabidopsis.org; verified 17 May 2009 Forceps
Lehle Seeds, provides seeds, literature, and useful information Arabidopsis wild-type strains Columbia (Col), Landsberg erecta
about Arabidopsis, and can be found at http://www.arabidopsis. (Ler), and Wassilewskija (Ws)
com; verified 17 May 2009 Arabidopsis mutant strain ttg and some other mutants selected by
the laboratory instructor
Nottingham Arabidopsis Stock Centre (UK), provides seeds
Selfed F1 seeds of a cross unknown to students
and information resources, and can be found at http://nasc.life.
Pots with soil
nott.ac.uk/; verified 17 May 2009

Description of the Exercise

In this exercise, you will be working on a mutant called
transparent testa glabra (hereafter ttg). This mutation
was generated following treatment of Wassilewskija (Ws)
seeds with ethyl methane sulfonate (EMS). The mutation
is caused by a single C-to-T base transition that introduces
a termination codon in place of the codon for the amino
acid glutamine 317 (Walker et al., 1999). In normal plants,
there are hair-like, specialized epidermal cells called tri-
chomes that are distributed on the leaves, stems, and sepals.
Trichomes are absent from the roots, hypocotyl, cotyledons,
petals, stamens, and carpels. The morphology of trichomes
varies from unbranched spikes to structures containing
two to five branches. You will study the trichomes in this
exercise. A number of genes are involved in the fate of the
trichomes: one group of genes initiates the formation of
trichomes and another group determines the shape of the
trichomes. In the ttg mutation, the trichomes are absent.
Check this phenotype in the procedure below. In addition
to the trichome phenotype, the ttg mutation eliminates the
purple anthocyanin pigments from the seed coat, causing
the yellow cotyledons to be visible through the testa. For
this reason, ttg seeds have a yellow color. Other seeds have
a brown color. Also, ttg mutants completely lack anthocya-
nins in the epidermis and in subepidermal layers of leaves
and stems. In genetics, mutant genes that exhibit several
and unrelated phenotypes are called pleiotropic. Therefore, Fig. 115. Wild-type and mutant trichomes in
ttg is a pleiotropic mutation. Figure 115 shows the shape Arabidopsis thaliana: (A) and (B) wild type, (C) dis-
of trichomes in the wild-type Columbia (Col) and a number torted 1 (dis1-1), (D) distorted 2 (dis2-1), (E) leaf of
the ttg mutation: note the lack of trichomes, and (F)
of mutant plants.
wurm-1 (wrm-1).

67 Genetics: A Laboratory Manual

Procedure
1. Study the three wild-type strains Col, Ler, and Ws.
Find similarities and differences between them. Note
that Ler is shorter than Col and Ws and has an upright
stem. Ws is an early flowering strain, and leaves are
slightly serrated and light-green in color. Also, check
the shape of the trichomes.
2. You may examine a number of mutant plants including
ttg. Examine each mutation in detail. Compare each
mutation with Col wild type and record the differences
on your report sheet.
3. You may set up a cross (see Protocol 112) between
two strains; for example, use ttg and Col with Col as
the male parent. Then collect the F1 seeds and plant
them in soil. You may allow the F1 plants to self-fertil-
ize to collect the F2 seeds. The F2 plants should show
you a 3:1 segregation of phenotypes. Alternatively, you
may back cross the F1 plants to the homozygous reces-
sive parent (in this experiment ttg/ttg) and check the
F2 progeny for a 1:1 phenotypic segregation. However,
with regard to the development time of Arabidopsis
and laboratory time restriction, your instructor may
give you the F2 seeds of a certain unknown (to stu-
dents) cross. These seeds will be the result of a cross
between ttg/Col ttg/Col, ttg/Col ttg/ttg, or ttg/Col
Col/Col plants.
4. Plant these seeds in pots. Cold treat them for 3 d at 4C,
and then transfer the pots to a greenhouse.
5. Forty-eight hours later seedlings should appear. Check
your pots regularly, add water as needed.
6. Two weeks later, bring the pots to the laboratory and
examine the F2 population for the presence or absence
of trichomes. Count the number of plants showing the
ttg phenotype and the number of plants showing the
wild-type phenotype. Do not discard the plants; you
will need them in Exercise 14; return the pots to the
laboratory instructor.
7. On the basis of your observations, answer the follow-
ing questions:
What are the phenotypes of the F2 plants?
What phenotypic ratio did you obtain?
Fig. 116. Corn cob bearing F2 kernels showing a 3:1
phenotypic segregation.
What type of cross are you dealing with, a self-cross or
a back cross? Explain why.
Which phenotype is recessive, which one is dominant?
Explain why.
Predict the genotype of the F1 and P plants.
8. Record your results in the table below. Fill out all col-
umns. The laboratory instructor will check your results
for accuracy.
Exercises with Corn (Zea mays)
Corn is one of the most important crop plants in the world.
Although corn genetics has been studied in detail, some
aspects of corn make it a difficult plant to use in laboratory
exercises; these include large size of individual plants and
long generation time. However, corn cobs (corn ears) are
ideal materials for studying transmission genetics.
An intact corn cob (Fig. 116) represents a population
of kernels. There are numerous genes scattered throughout
the corn genome that determine the color, shape, and other
characteristics of the kernels. One advantage of using corn
in genetic studies is the variety of genetic interactions that
exist among its genes. For example, if two independently
segregating genes both affect the same phenotype, then the
classical 9:3:3:1 ratio will be changed depending on the
genes involved and the type of the interaction. Such inter-
actions are called epistasis. The gene whose phenotype
is expressed, is called epistatic, whereas the gene whose
phenotype is masked (suppressed), is called hypostatic.

Observed (O) Expected (E)

number of number of
Seed code seedlings per seedlings per
(if any) phenotype phenotype OE (O E)2/E 2 df

Gregore Koliantz & D.B. Szymanski 68

 Observed (O) Expected (E)
number of number of
Cob code Kernel pheno- kernels per kernels per
(if any) type phenotype phenotype
Variants of the standard 9:3:3:1 ratio, are described below.
12:3:1 One dominant gene masks the expression of the
other gene.
9:3:4 Homozygous recessive alleles mask the expression of
one of the dominant alleles.
9:6:1 Each dominant gene alone, but not in combination, pro-
duces the same phenotype.
15:1 Each dominant gene alone or combined shows the
same phenotype.
13:3 The dominant allele of one of the two genes masks
the expression of the other dominant gene and the double-
recessive alleles.
9:7 Both dominant genes are required for the expression of
a functional product; under this condition, each allelic pair
would result in a nonfunctional (mutant) phenotype; this inter-
action is sometimes called a complementary gene action.
Description of the Exercise
In this exercise, you will examine one or more corn cobs by
studying kernel color and texture. You have to collect data
on the number of kernels showing variety of phenotypes
and find out what cross is represented by the corn.
Materials
Corn cobs representing the F2 population of unknown
crosses. The cross could be a monohybrid, a dihybrid, or a
back cross. The laboratory instructor will assign corn cobs
to students.
Procedure
1. Pick a corn cob.
2. Examine the phenotype of kernels. Count the number
of kernels belonging to each phenotypic group.
3. From the results, predict the type of cross, the genotype
of the P, the F1, and the F2 cobs.
4. Test your conclusions by the Chi-square test.
5. Record your observations in the table at the top of this
page. Fill out all columns. The laboratory instructor
will check your results for accuracy.
Sex-Linked Inheritance
Sex chromosomes are a pair of heteromorphic (morpho-
logically different) or homomorphic (morphologically
identical) chromosomes that determine sexes in organisms.
Sex-linked inheritance is a pattern of transmission of genes
that are located on the sex chromosomes.
69
OE (O E)2/E 2 df
Almost all animal species have a pair of sex chromo-
somes. In some, but not all, dioecious plants (having
pistillate and staminate flowers on different individuals)
sex chromosomes have been identified. Spinach (Spinacia
oleracea L.) and bladder campion (Silene latifolia Poir.)
have heteromorphic sex chromosomes, whereas those in
papaya (Carcia papaya L.) are homomorphic. Monoecious
plants (having pistillate and staminate flowers on the same
individuals) and hermaphrodites (flowers on the same indi-
viduals have both male and female reproductive organs) do
not have sex chromosomes.
In the exercise described below, you will study the sex-
linked inheritance in the fruit fly Drosophila melanogaster.
Exercises with Drosophila melanogaster
The fruit fly, Drosophila melanogaster (hereafter called
Drosophila, unless otherwise specified), is the most exten-
sively studied organism in the animal kingdom. It belongs to
the phylum Arthropoda; class Insecta; order Diptera; family
Drosophilidae; genus Drosophila; and species melanogaster.
Drosophila is cosmopolitan: it is found all over the world. It
lives and breeds in decaying fruits and flowers. The two spe-
cies melanogaster and simulans are in close association with
humans and are referred to as domestic species.
Drosophila was first studied by W.E. Castle in 1901 as
an experimental organism, and then it was used by T.H.
Morgan for genetic experiments. In 1910, he proposed the
theory of sex-linked inheritance, and the following year,
C.B. Bridges discovered that sex-linked genes are located
on the X chromosome. In 1913 A.H. Sturtevant determined
that genes are arranged on the chromosomes in a linear
order, consequently he prepared the first genetic map of
Drosophila. In 1927, H.J. Mueller discovered that X-rays
can cause lethal mutations and can increase the frequency
of spontaneous mutations in Drosophila. In 1935, C.B.
Bridges successfully prepared the first map of Drosophila
polytene chromosomes. The first catalogue of Drosophila
mutations was prepared in 1944 by C.B. Bridges and K.S.
Brehme. Twenty-four years later, a more comprehensive
catalogue was introduced by D.L. Lindsley and E.H. Grell.
The updated version of this work appeared in 1992 edited
by D.L. Lindsley and G.G. Zimm. Further revisions and
additions to this catalogue are published electronically
Genetics: A Laboratory Manual
and can be found in Drosophila resource sites. In 1950,
M. Demerec published the first book on the biology of
Drosophila. A complete review of the genetics and biol-
ogy of Drosophila was published in 12 volumes between
1976 and 1986, edited by M. Ashburner, E. Novitski, T.R.F.
Wright, H.L. Carson, and J.N. Thompson, Jr. Drosophila
Information Service (known as DIS) presents informal
reports and information from Drosophila workers around
the world and is published annually.
Life Cycle
The life cycle (development time) of Drosophila varies
depending on the culture temperature. The development
time of wild-type strains is about 18 d at 18C, around 11
d at 25C, and close to 9 d at 29C. Mutants have a longer
development time.
In the life cycle of Drosophila, four distinct stages can
be identified: egg, larva, pupa, and adult (Fig. 117). At
25C, about 24 h after insemination, eggs are laid by the
female parent. A healthy female can lay eggs for 6 to 8 con-
secutive days. The day after the eggs are laid, larvae hatch;
they are called the first instar larvae. In the next 4 to 5 d, the
larvae molt twice, producing the second and the third instar
larvae. During molting, the larvae undergo a considerable
increase in size, exclusively because of increase in cell size.
Fig. 117. The Drosophila melanogaster life cycle. For
details see the text.
Gregore Koliantz & D.B. Szymanski
However, the cells of the imaginal discs which will form
adult structures during the pupal stage, do divide during
this period. Eventually, the cuticle of the third instar larvae
hardens to become the pupae. Metamorphosis occurs within
the pupae. During metamorphosis, all larval cells except
those of the Malpighian tubules and the nervous system
degenerate and adult structures are formed by the growth
and morphogenetic movements of the imaginal disc cells.
The pupal stage takes about 5 d. When the metamorphosis
is complete, the adults emerge (eclose) through the anterior
end of the pupae. Upon emergence, the males are sexually
mature, but the females remain unreceptive to the males for
about 8 h. During this time, the females remain virgin.
Model Organism
Drosophila is widely used in genetic experiments for the
following reasons:
it is relatively small in size
sexes are easily distinguishable
it reproduces easily and can be grown in the laboratory without
the need of expensive equipment
it has a relatively small genome which has been sequenced
it has polytene chromosomes which can be used to identify
gene loci and chromosomal rearrangements
a large number of mutations have been isolated and characterized
high level of homology has been found between the Drosophila
and human genome
The Genome
Drosophila is a diploid fly with a diploid number of 8 chro-
mosomes (2n = 8). The entire genome was sequenced in
1999 and some 13,600 genes were identified. This number
is now 30,887 released by the Genomic Center of Indiana
University in 2007 (http://flybase.org/static_pages/docs/
release_notes.html; verified 17 May 2009).
Wild-Type and Mutant Strains
Wild type is referred to the flies which do not carry
mutations. The most commonly used wild-type stock
is Oregon-R, but Canton-S, Samarkand, and a few more
stocks are used in many laboratories. The list of mutations
discovered and characterized so far can be found on the
websites of Drosophila resource centers (see below). Table
115 lists a variety of Drosophila mutants widely used in
the laboratory.
Selected National and International Resources
A wealth of information about Drosophila is available in
the following sites:
Indiana University Genomic Information for Eukaryotic
Organisms; found at http://eugenes.org; verified 17 May 2009
Indiana University Database of Drosophila Genes and Genomes:
the electron micrograph maps of polytene chromosomes (Sorsa
Maps); found at http://flybase.bio.indiana.edu/static_pages/allied-
data/external_resources5.html; verified 17 May 2009
Berkeley Drosophila Genome Project (BDGP); found at
http://www.fruitfly.org; verified 17 May 2009
Cambridge University (UK) Database for Drosophila and
Anopheles Genomics; found at http://flymine.org; verified 17
May 2009
70
Table 115. Representative of Drosophila melanogaster mutations. carrying one or two additional
Y chromosomes, (2A:XYY or
Gene Map 2A:XYYY, respectively) are
symbol Name Phenotype Chromosome position viable but sterile too. These
cM observations indicate that there
B Bar Eyes narrow X 57.0 are a number of male fertility
w white Eye color white X 1.5 genes on the Y chromosome and
sn singed Bristles short and deformed X 21.0 that excess of the fertility genes
vg vestigial Wings reduced to vestiges 2 67.0
bw brown Eye color light brown darkening with age 2 104.5 may change the genic balance,
Cy Curly Wings curled upward 2 6.1 so the males become sterile. On
e ebony Body color shining black 3 70.7 the other hand, females carrying
Sb Stubble Bristles less than one-half normal length 3 58.22 three X chromosomes (2A:XXX)
ey eyeless Eye size reduced 4 2.0 are phenotypically female but
bt bent Wings bent sharply backward 4 1.4 survive only a few hours after
gy goutylegs Legs shortened and thickened 4 0.2
emergence from pupae. They
Semi dominant; eyes oval in females. are called metafemales (Stern,
Dominant, homozygous lethal. 1959). Presence of an additional
set of autosomes in flies destabi-
lizes sexes, shifting the females
The Chromosomal Basis toward an intersex phase and
of Sex Determination the males to a metamale (Stern, 1959) state. Metamales are
In Drosophila, chromosomal differences are visible between relatively inviable, sterile flies and show a delayed devel-
sexes: the males are heteromorphic, carrying one X and one opment time. Thus, in Drosophila, sex is determined by a
Y chromosome, whereas the females are homomorphic, balance between genes that are located on the X chromo-
having two X chromosomes ( Fig. 118). Chromosomes some and the autosomes (Table 116).
2, 3, and 4 are the autosomes. During meiosis, the sex
chromosomes segregate ensuring a 1:1 ratio of males and Description of the Exercise
females, as illustrated in Fig. 119. In this exercise, you will study the inheritance of the
The Y chromosome does not have a role in sex deter- characters associated with the X chromosome of D. mela-
mination or the viability of Drosophila. Flies with two sets nogaster. You will set up crosses between a wild-type and
of autosomes and one X chromosome (2A:X) are viable, a mutant strain. Then you will examine the progeny of each
phenotypically male but sterile. Furthermore, normal males cross to study the pattern of the inheritance. The laboratory
instructor may give you advice in choosing the sex-linked
mutations. More information about the mutations can be
found in Lindsley and Zimm (1992).
Materials
Drosophila stocks
wild type: Oregon-R
mutants:
white; compound eyes and the ocelli white, also no
pigments in larval Malpighian tubules and adult
testis sheath
miniature; wing size reduced and less transparent
Fig. 118. A schematic representation of Drosophila than the wild type
melanogaster mitotic chromosomes; (a) in females, (b) Drosophila culture medium in vials (see Protocol 116 for details)
in males. Dissecting microscope or powerful magnifying glass

Table 116. The relationship between chromosome num-

ber and sex determination in Drosophila melanogaster.

No. of No. of
autosomal set X chromosomes
(A) (X) Ratio A/X Sex
2 1 2 normal male
2 2 1 normal female
2 3 0.67 metafemale
Fig. 119. Segregation of the sex chromosomes in 3 2 1.5 intersex
Drosophila melanogaster. Autosomes are not shown 3 1 3 metamale
for simplicity. Expression of the characters of both sexes.

71 Genetics: A Laboratory Manual

Sorting plates
Sorting brushes
Drosophila anesthetizer, (Carolina Biological Supply Co.,
Burlington, NC)
Ether (ethyl ether)
Glass or polystyrene vials
Morgue: jar containing alcohol or a detergent
Incubator adjusted to 25C

Procedure
A. How to Anesthetize Flies
Caution: You will be working with ether! Ether is highly
flammable and is harmful to your health.
1. The Drosophila anesthetizer is composed of a chamber,
a hollow stopper on the mouth of the chamber, and a
cap in the bottom. A foam pad has been installed in the
bottom of the chamber (Fig. 1110).
2. Remove the hollow stopper from the mouth of the
chamber and fill it one-third full with ether. Do not ap-
ply more ether because the flies will be killed.
3. Remove the cap to expose the foam pad, then quickly
pour the ether on the pad. Replace the cap and the stop-
per securely. The anesthetizer is now ready to be used.
4. To anesthetize flies, remove the stopper; at the same
time gently shake down the Drosophila culture vial,
quickly unplug the vial and put the mouth of the
vial on the mouth of the anesthetizing chamber; tap
gently on the vial to shake the flies from the vial
into the chamber.
5. Remove the culture vial and plug it; then immediately
put the stopper back on the mouth of the chamber and
tap the bottom of the anesthetizer on the bench to push
the flies to the bottom of the chamber.
6. The flies should stop moving in about 20 to 30 seconds
at room temperature. Remove the flies for examination
and sorting. The flies remain anesthetized usually for
8 to 10 min. At temperatures higher than 25C, they
recover quickly.
B. Identify Body Parts
1. You will be given live flies in a clean vial.
2. Anesthetize the flies as described above.
3. Place the anesthetized flies on a sorting plate and iden-
tify body parts. Use Fig. 117 as a guide. Touch the
flies with brush.
C. Identify Sexes
Females are larger than the males; well-fed and healthy
females measure about 3 mm and the males 2 mm.
In females, the abdomen is large and the posterior end is
pointed; in males, the abdomen is short and round.
Alternating dark-light chitinous bands (tergites) on
the dorsal end of the abdomen are visible in females,
whereas in males, the last few tergites are fused to-
gether to form a large, dark band.
Check the differences in the external genitalia on the
ventral end of the abdomen of each sex.
On males there are stiff hairs (chaetae) on the first tarsal
segment of each foreleg. This is called sex comb.
Fig. 1110. The Drosophila anesthetizer. For details see
the text.
D. Set up Crosses
1. Set up a reciprocal cross between the wild-type and the
mutant (white or miniature) flies as shown below. Note
that these mutations are located on the X chromosome.
Make sure that the female flies you will choose are
virgin (see Protocol 114 for collecting virgins).
Otherwise, the laboratory instructor may give you vir-
gin females.
Cross 1 Cross 2
Female Male Female Male
P Wild type mutant mutant Wild type
F1 ? ?
2. Anesthetize the flies and identify sexes.
3. With the aid of a brush, transfer five females and the
same number of males onto the glass surface of a vial
containing culture medium. Plug the vial. Do not drop
the anesthetized flies onto the surface of the me-
dium: they will stick to the medium and will die! Do
not stand the vial on end until the flies have revived.
4. Place the vial in a 25C incubator.
5. One week after the cross has started, remove the parents
from the vials (drop them into the morgue) to ensure
that only the progeny will remain in the vials. Check
the medium attentively; the larvae should be visible at
this time. Leave the vial in the same incubator for one
more week.
6. In the time specified, bring the vials to the laboratory;
you will see a number of adult flies in the medium;
they are the F1 generation of the cross you set up 2 wk
ago. Anesthetize the flies and check the phenotype of
the males and the females separately.
7. On the basis of your observations, answer the follow-
ing questions:

Gregore Koliantz & D.B. Szymanski 72

 What were the phenotypes of the parents? 7. Just before planting the seeds, spray water on top of the
pots to keep the soil moist.
8. After seeds have been planted, cold treat the pots in a
What were the phenotypes of the progeny? 4C refrigerator or cold room for 3 d to synchronize
seed germination.
9. Transfer the pots to a greenhouse or to a growth cham-
Did you see phenotypic difference between the males ber, if available.
and the females in the F1 generation? Describe and dis- 10. Forty-eight hours later, remove the dome and let the
cuss your observations. seedlings grow. Water the plants regularly, but do not
over-water them. Occasionally use fertilizer when wa-
tering the plants. Mix 1/2 teaspoon of the fertilizer of
What was the sex ratio in the F1 flies? Did the experi- choice with 1 L of water.
ment confirm your expectation? In optimal conditions, after a period of about 6 wk,
siliques start to form and eventually they turn brown (yel-
low in an extended amount of time). Stop watering the
Use the Chi-square test if needed. plants when the siliques turn brown.
To collect seeds:
1. Hold the plant horizontally above a clean and dry piece
Protocol 111 of paper (white paper is preferred because brown seeds
Preparation of Pots for Planting will be easily visible).
Arabidopsis Seeds 2. Gently rub the plant so that the siliques shatter and the
Arabidopsis can be grown in a variety of containers includ- seeds fall onto the white paper.
ing pots and trays (flats). Here we describe preparation of 3. Remove excess dry matter such as dry leaves, shoots,
pots for laboratory use. etc., by hand and separate the seeds from the chaff with
fine forceps.
Materials 4. Collect seeds in microtubes (conical tubes in case a
Pots, usually 5 5 cm large quantity of seeds are harvested), then add two to
Tray (flats), with and without holes three pellets of anhydrous calcium sulfate to each tube
Plastic domes to fit the flats to dehumidify the seeds. The pellets are blue in color;
Soil: Scotts Redi-earth (The Scotts Company ) or equivalent
they turn pink when they absorb moisture. Replace the
Vermiculite, extra coarse: Sunshine (Sun Gro Horticulture)
or equivalent pellets as needed.
Larvicides/insecticides for basic pest control: Note: Preparation of flats is almost the same as that of pots.
Gnatrol (Valent Professional Products) or equivalent, to 1. Place a tray with holes into the tray without holes.
kill larvae in soil 2. Add 10 mL of a larvicide to 4 L of water, mix well,
Marathon (Olympic Horticultural Products) or equivalent, then pour the water into the tray.
to kill common pests such as thrips 3. Fill the tray one-half full with extra-coarse vermiculite.
Fertilizer: 4. Mix 1/2 teaspoon of a pesticide with 4 L of soil.
Miracle-Gro (Scotts Miracle-Gro Products, Inc.) or equivalent 5. Add the soil (prepared in Step 4, above) on top of the
Anhydrous calcium sulfate pellets: Drierite (W. A. Hammond
vermiculite.
DRIERITE Co. LTD) or equivalent, to dehumidify
seeds 6. Cover the tray with a plastic dome and keep at room
Deionized water (preferred), otherwise tap water temperature until use.
Graduated cylinders 7. Just before planting the seeds, spray water on top of the
Seeds of interest soil to keep it moist.
Sheets of clean and dry paper
Protocol 112
Procedure
1. Place a tray with holes into the tray without holes, then
Setting up Arabidopsis Crosses
Arabidopsis thaliana is a self-fertilizing plant; that is, it
put pots in it.
carries both male and female reproductive organs (see Fig.
2. Add 10 mL of the larvicide of choice to 4 L of water,
114B). The male reproductive organ is the stamen, which
mix well, then pour the water into the tray, so that the
consists of an anther and filament. Anthers carry pollen
pot will be half-filled.
grains: the male germ cells. The female reproductive organ
3. Fill each pot two-thirds full with extra-coarse vermiculite.
is the carpel, which is made up of stigma, style, and ovary.
4. Mix 1/2 teaspoon of the pesticide of choice with 4 L
Eggs develop in the ovary. During self-fertilization, pollen
of soil.
grains adhere to the stigma and germinate. Then the pollen
5. Fill each pot with the soil mentioned in Step 4.
tube grows through the ovarys tissues, penetrates the ovule
6. Cover the pots with a plastic dome and keep in room
to fertilize the egg. In the laboratory, it is possible to cross-
temperature until needed.
fertilize Arabidopsis.

73 Genetics: A Laboratory Manual

Materials in a dry place. These seeds are the F1 of the parents you
Plants of interest used for the cross.
Dissecting microscope
Forceps Protocol 113
Small scissors
Microtubes, 1.5 mL
Ethylmethane Sulfonate Mutagenesis of
Petri dish Arabidopsis Seeds
Anhydrous calcium sulfate pellets (see Protocol 111 for details) Mutagens are valuable tools in studying the function of
Aluminum foil genes in living organisms. Arabidopsis can be mutagenized
Plastic-film wrap by alkylating agents, irradiation, or T-DNA insertion. One
Plastic pail of the widely used chemical mutagens is methanesulfonic
Ambient-controlled incubator or greenhouse
acid ethyl ester, generally known as ethylmethane sulfonate
Procedure or EMS. EMS is a colorless alkylating agent with a molecu-
Practically, plants of any age can be used for genetic crosses, lar weight of 124.1142 and the linear formula CH3SO3C2H5.
as long as they have buds and open flowers. However, the EMS induces point mutations in DNA in the form of a GC
best result can be obtained if plants of interest are at the to AT, or AT to GC transition.
primary inflorescence stage. Perform crosses under a dis- Caution: Studies have shown that EMS causes cancer
secting microscope. in animals. In humans, it causes potential health effects
1. Choose a cluster of buds that are at the tip of the primary on eyes and skin, as well as irritation of the digestive and
inflorescence. Pick a bud whose sepals are barely visible respiratory tract. Therefore, work with EMS must be done
from the topmost point of the operculum. At this stage, under a fume hood and gloves must be worn throughout
the stigma is mature enough to take pollen grains. the experiment.
2. With forceps or small scissors, and under a dissecting Materials
microscope, remove any bud from the cluster that is EMS (ethylmethane sulfonate)
not going to be emasculated. Leave one or preferably 0.1% Triton X-100 (t-octylphenoxypolyethoxyethanol; Sigma, St.
two buds for emasculation. Louis, MO)
3. With the aid of forceps, open the bud of interest and Per 100 mL of dH2O
100 L Triton X-100
carefully remove the pollen sacs. Note that Arabidopsis
Mix well
has six anthers, each carrying a single pollen sac. It 100 mM sodium thiosulfate:
is not necessary to cut the entire anther. During emas- Per 500 mL of dH2O
culation, care must be taken not to damage the style. 12.41 g Sodium thiosulfate
4. Once the pollen sacs have been removed, the bud serves Mix well
as the female parent and can be used for pollination. 0.1% agar:
5. The plant from which pollen grains are taken (the donor Per 1000 mL of dH2O
plant), serves as the male parent. With forceps, detach a 1 g Agar
fully opened flower from the male parent, remove one Autoclave
Double-distilled water
anther or two, then rub the stigma of the female parent
Seeds of interest
(Step 4, above) with the anther. Make sure enough Anhydrous calcium sulfate pellets (see Protocol 111 for details)
pollen grains are accumulated on top of the stigma. Micropipettors
6. Tag the inflorescence which carries the pollinated bud Tips
with a strip of aluminum foil. Be sure not to damage Dissecting microscope
the inflorescence. Tagging will help you quickly and Microtubes, 1.5 mL
correctly identify the bud you used for the cross. Glassware
7. When the cross is complete, place the plant into a Petri Transfer pipettes
dish which is filled with water and immediately trans- Conical tubes
Conical tube racks
fer the plant into an ambient-controlled incubator that
Whatman No. 1 paper
has a 24-h light cycle. If the cross has been successful, Platform mixer
pod elongation will start in 2 to 3 d. Alternatively, if Flats of soil (see Protocol 111 for details)
you are using a greenhouse or light racks, place the Fume hood
plant in a plastic pail with some water in it, cover the Ambient-controlled incubator or greenhouse
pail with plastic-film wrap, and put it in a green house
Procedure
under continuous light. As soon as pod elongation
1. Weigh 1 g of seeds of interest.
starts, remove the plant from the pail and place it on
Note: One thousand Arabidopsis Ler seeds weigh about
the rack.
20 mg; therefore, 1 g would be about 50,000 seeds. Also,
8. Harvest the seeds from the pod when it turns brown.
the volume of 1 g of seeds is about 1.8 mL.
Collect seeds into a microtube, put a small pellet of
2. Resuspend the seeds in 0.1% Triton X-100 using a
anhydrous calcium sulfate in the microtube, and save it
conical tube.

Gregore Koliantz & D.B. Szymanski 74

3. Tape the tube on a platform mixer and mix the tube
overnight (usually 12 h).
4. Wash the seeds five times in double-distilled water.
After the last wash, let the tube sit undisturbed for 10
to 15 min. Then with a transfer pipette, carefully trans-
fer all the water above seed level. Read the volume of
the wet seeds. Subtract that volume from 1.8 (that is
the volume of 1 g of dry seeds). The outcome is the
volume of the water present in the tube.
5. Wear gloves and take the tube (from Step 4, above)
to a fume hood. Add distilled water to the tube to a
total volume of 20 mL. Then add 60 L of EMS to the
tube. The final concentration of EMS is now 0.3%. The
concentration can be changed as needed.
6. Secure the cap of the tube, tape the tube on a platform
mixer, and mix for 14 h at room temperature.
7. Decant (as much as possible) the EMS solution into a
beaker containing 100 mM sodium thiosulfate to deac-
tivate the EMS.
8. Wash the seeds twice in 100 mM sodium thiosulfate,
15 min each.
9. Decant (as much as possible) the sodium thiosulfate,
then thoroughly wash the seeds with double-distilled
water five times.
10. After the last wash, decant the water containing the
seeds onto a large piece of Whatman paper and let the
seeds dry for several hours.
11. Collect the seeds into a conical tube, add two to three pel-
lets of anhydrous calcium sulfate, and save for future use.
To plant mutagenized seeds:
1. Place mutagenized seeds in 1.5-mL microtubes, fill the
tubes with double-distilled water, and cold-treat the
seeds at 4C for 3 d.
2. With transfer pipettes, transfer the cold-treated seeds to
2 L of 0.1% agar solution.
3. Sprinkle 100 mL of the seed solution in each flat of
soil, that is, 5000 seeds per flat. Therefore, 10 flats of
soil are needed to plant the mutagenized seeds.
4. Cover the flats with a dome, transfer them to an
ambient-controlled incubator or green house, let
the seeds germinate at 100% relative humidity for
3 to 4 d, then remove the dome. The seedlings are
the M1 population.
5. Let the M1 population self-fertilize and then collect the
seeds. Call the seeds M2.
6. Plant the M2 seeds in flats, let the seeds germinate, then
screen the M2 seedlings under a dissecting microscope
for altered phenotypes.
7. If a mutant seedling is found, remove the seedling from
the soil and replant it in a pot. Save the putative mutant
plant for further studies.
Protocol 114
How to Maintain Drosophila Stocks
The only way to maintain Drosophila stocks is to transfer
the flies to fresh medium every two weeks if raised at 25C,
or every three to four weeks if kept at 18C.
75
To transfer the flies from an old culture vial to a fresh
medium, remove the plug from the fresh vial; at the same
time gently tap the bottom of the culture vial against the
palm of your hand to send the flies to the bottom of the
vial; then quickly remove the plug and invert the vial over
the mouth of the fresh vial. Shake in 5 to 10 flies from each
sex and recap both vials. Properly label fresh vials with the
name of the mutation and the date of transfer. Time to time
check the phenotype of the flies to ensure that they are not
contaminated with other stocks. Wash old vials with deter-
gent and autoclave for further use.
Protocol 115
How to Collect Drosophila Virgin Females
In order to set up crosses between flies of different geno-
types, it is extremely important to use virgin females. This
is because females already inseminated by a male can store
the sperm in the spermatheca (sperm storage sac in the
female reproductive tract) and use it for up to six days.
Therefore, to be sure that the desired gene was involved in
a cross, the female parent must be virgin.
The simplest method to collect virgin females is to
remove adult flies from the culture vial, place the vial in a
25C incubator, and collect all those females that emerge in
the next 6 to 7 hours. Keeping the vials at 18C will reduce
the rate of eclosion, but the females eclosing in the next 12
hours will most likely be virgin. In cultures which contain
adult flies of different ages, virgin females can be identi-
fied by light body color or especially by the presence of the
dark meconium (the first stool) in the gut. You can see the
meconium in the ventral region of the abdomen.
Protocol 116
How to Prepare Drosophila Medium
Regular Medium
Traditionally, regular medium is referred to the medium
which is cooked in the laboratory. Variety of recipes and
modifications of a standard recipe are available, but just
which is used depends on the availability of the raw materi-
als and the laboratory temperament. Here we describe the
medium we use in our laboratory to maintain the stocks.
Materials
Per 1000 mL of deionized water
50 g Cornmeal
32 g Granulated sugar (edible sugar)
50 g Bakers yeast
11 g Agar
Note: The amount of the agar must be adjusted as different
brands of bakers yeast may affect the firmness of the
medium.
10% Nipagin M [methyl paraben (methyl ester of p-hydroxyben-
zoic acid) Sigma, St. Louis, MO]
Add 10 g of Nipagin M to 100 mL of ethanol, mix well, and
use as stock. Nopagin M is a mold inhibitor.
Autoclaved deionized water
50% bleach
Equal volumes of bleach and deionized water
Ethanol
Genetics: A Laboratory Manual
Vials measuring 3 10 cm (diameter height), commercially
known as Drosophila vials; borosilicate glass (preferred)
or polypropylene
Nonabsorbent cotton wool or polyurethane foam plugs to fit the vials
Drosophila culture bottles, 240 mL (Carolina Biological Supply
Co., Burlington, NC) with polyurethane foam plugs
Cheese cloth
Saucepan
Range
Procedure
Before preparing the medium, autoclave culture bottles and
glass vials. If using polypropylene vials, wash them with
50% bleach and rinse in autoclaved water.
1. In a saucepan mix cornmeal, sugar, bakers yeast, and
agar, add water and bring to the boil while stirring con-
stantly to avoid lumps.
2. Let the mixture boil and simmer for 10 min.
3. Add 16 mL of 10% Nipagin M; stir well, and distribute
the medium in sterile vials to a depth of 3 cm or in
sterile culture bottles to a depth of 2 cm.
4. Cover the vials or bottles with a sheath of clean cheese
cloth and leave for 2 h.
5. Plug the vials or bottles with polyurethane foam plugs
or nonabsorbent cotton wool. Foam plugs are preferred
because they can be washed, autoclaved, and reused
for an extended amount of time.
6. Store the vials and bottles in a refrigerator.
7. Just before use, allow the media to warm to room
temperature.
Recommendations
We recommend the following points when preparing the
regular medium.
1. In this medium, bakers yeast is superior to brewers yeast.
2. Drosophila larvae burrow into the medium to the depth
of about 10 mm; therefore, increasing the depth of the
medium will not improve the larval growth. Expanding
the area of the medium will increase larval viability
and weight, hence decreasing the mortality.
3. It is unnecessary to seed the medium with additional
live yeast. However, for extremely weak mutants,
some 10 grains of active dry yeast can be added to the
medium in each vial at the time when larvae are at the
first instar stage. For the bottles, some 30 grains would
be sufficient.
Instant Medium
The instant medium (Formula 4-24, by Carolina Biological
Supply Co., Burlington, NC) is available as a powder, ready-
to-use medium. According to the manufacturer, it is protected
with anti-oxidant and mold inhibitor. We use this medium for
class exercises.
Materials
Formula 424 Instant Drosophila Medium
Active dry yeast (available in grocery stores)
Autoclaved distilled water
Vials measuring 3 10 cm (diameter height), commercially
known as Drosophila vials; borosilicate glass (preferred)
or polypropylene
Gregore Koliantz & D.B. Szymanski
Measuring spoons
Nonabsorbent cotton wool or polyurethane foam plugs to fit the vials
Procedure
Use autoclaved or sterilized vials as described above.
1. To the vials measuring 3 10 cm, add 10 mL of the
instant medium and an equal amount of autoclaved,
distilled water. Sprinkle about 10 grains of active dry
yeast on top of the medium.
2. Plug the vials with nonabsorbent cotton wool or poly-
urethane foam plugs, let set for 30 min before use.
Study Questions
111. In garden pea, tall stem (T) is dominant to short
stem (t), green siliques (G) are dominant to yellow
siliques (g), and smooth seeds (S) are dominant
to wrinkled seeds (s). The genes controlling these
phenotypes are independently segregating. In a cross
between homozygous tall, green, smooth and short,
yellow, wrinkled plants:
a. What will be the phenotype of the F1 plants?
b. What will be the phenotype of the F2 plants?
c. What will be the phenotype of the F1 back cross to
its homozygous recessive parent?
112. How many genetically different pollen grains could be
formed in Arabidopsis with the following genotypes:
a. aa Bb CC Dd
b. Aa Bb Cc Dd
113. In Drosophila melanogaster, flies with the sex chro-
mosome complement XY or XO are males and those
with XX, XXX, or XXY are females. What is the role
of the Y chromosome in sex determination?
114. In a cross between two plants both of which express
tall stem and red flowers: 238 tall and red, 89 tall
and white, 78 short and red, and 27 short and white
offspring were obtained. What are the genotypes of the
parent plants? Assume tall and red are dominant genes.
115. How do monoecious plants differ from dioecious
plants? Is Arabidopsis thaliana a monoecious, dioe-
cious, or hermaphrodite plant? Explain your answer.
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