Practical Of Genetics
BIOL 2121
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To prepare slides of onion root tips
demonstrating the stages of mitosis (somatic
cell division) and identification of cells in the
various stages of mitosis.
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Prokaryotic cells (bacteria) reproduce asexually
by binary fission. Bacterial cells have a single
circular chromosome, which is not enclosed by a
nuclear envelope.
During binary fission the bacterial chromosome
is duplicated, the cell elongates, and the two
chromosomes migrate to opposite ends of the
cell.
Each daughter cell receives one chromosome
and is identical to the parent cell.
Binary fission is a relatively fast and simple
process.
Eukaryotes are diploid, which means they have
two sets of chromosomes; one set of
chromosomes is inherited from each parent.
 For example in a growing plant root, the cells at
the tip of the root are constantly dividing to
allow the root to grow.
 Because each cell divides independently of the
others, a root tip contains cells at different
stages of the cell cycle.
 This makes a root tip an excellent tissue to study
the stages of cell division.
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The cell division cycle : is a sequence of events in a
eukaryotic cell between one mitotic division and
another.
 It includes :
1. Interphase (G1 phase, S phase, and G2 phase)
2. M phase. In the M-phase both the nucleus and
the cytoplasm divide (mitosis and cytokinesis).
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Chromosomes are visible only during mitosis when
they separate between the daughter cells.
 The process of mitotic division ensures that both
daughter cells will gain identical set of chromosomes
containing identical genetic information.
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It takes about 16 hours
Interphase
Mitosis
G1
S
G2
M
5
7
3
1
Vary among cell types
Consistent among cell types
Duration of phases of Mitosis:
Prophase: 36 minutes
Metaphase: 3 minutes
Anaphase:
3 minutes
Telophase: 18 minutes
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Chromatin
appears
dispersed,
DNA
replication
occurs.
(Chromatin is a mass of uncoiled DNA and
associated proteins called histones).
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Mitosis, or nucleus division, is the first part of
M-phase and in consists of four stages
(prophase, metaphase, anaphase and
telophase).
1. Prophase:
 Chromatin condenses
 chromosomes become visible
 nuclear envelope and nucleoli disappear
 spindle starts to form attach to the
kinetochore ( a portion of the centromere).
 Each replicated chromosome comprises two
chromatids, both with the same genetic
information (called sister chromatids).
Prometaphase:
 In this stage the nuclear envelope breaks
down so there is no longer a recognizable
nucleus.
 Some mitotic spindle fibers (microtubules)
elongate from the centrosomes and attach to
kinetochores (protein bundles at the
centromere region on the chromosomes
where sister chromatids are joined).
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These microtubules which attach to the
kinetochore
are
called
“kinetochore
microtubules”.
Other spindle fibers elongate but instead of
attaching to kinetochores, they attach with
spindle fibers growing from the other side of
the cell. These are called “polar microtubules
or non-kinetochore microtubules”.
The chromosomes start to migrate towards
the center of the cell by the kinetochore
microtubules
2. Metaphase
 All chromosomes align in one plane at the
center of the cell called the equatorial plane
(also referred to as the metaphase plate).
Anaphase:
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Spindle fibers shorten
the kinetochores separate
the
sister
chromatids
(daughter
chromosomes) are pulled apart and begin
moving to the cell poles.
Telophase:
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Telophase, the last stage of division, is
marked by a noticeable condensation of the
chromosomes, followed by formation of a
new nuclear envelope around each group of
chromosomes.
The chromosomes gradually de-condense to
form the chromatin seen in interphase. The
nucleoli reappear.
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Usually occurs at the end of telophase.
In plant cells cytokinesis is accomplished by
the formation of a cell plate.
Animal cells separate by forming a cleavage
furrow.
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It is possible for you to make your own
stained preparations of onion root tips and
observe mitotic figures (2n=16).
Onion bulbs have been rooted in water.
Growth of new roots is due to the production
and elongation of new cells.
Mitotic divisions are usually confined to the
cells near the tip of the root.
Follow the procedure outlined below to make
your own root tip preparation.
The roots are easy to grow in large numbers.
The cells at the tip of the roots are actively
dividing, and thus many cells will be in stages
of mitosis.
3. The tips can be prepared in a way that allows
them to be flattened on microscopes slide
(“squashed”) so that the chromosomes of
individual cells can be observed.
4. The chromosomes can be stained to make
them more easily.
1.
2.
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Onion bulb with roots
Crystal violet and other stains.
Fixative solution (1 part glacial acetic acid to 3 parts
ethanol).
1M HCl
Clean slide & cover
Forceps
Razor blade
Fingernail polish
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1. Obtain two small cups, label one “ HCL”
and the other “Carnoy.” Pour in enough of
each to just cover the bottom of its cup.
2. Obtain an onion bulb that is just beginning
to show the emergence of roots. Cut a small
piece of root off and use forceps to place it in
the cup of 1M HCL for 4 min at 60ºC.
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3. Transfer the root into the cup of carnoy
fixative. The root should remain in the carnoy
fixative for 4 minutes.
4. After the 4 minutes, place the root on a
slide. Then, cut off the bottom 1 or 2mm of
the root tip & discard the rest.
5. Cover the root with a 1drop of Crystal
Violet for 2 minutes. After the 2 minutes, blot
away the stain; be careful not to touch the
root tip.
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6. When the stain has been blotted away, cover
the root tips with drops of water to remove the
stain. Apply water drops until water runs mostly
clear. Blot slide without touching root tip.
7. Gently lower a cover slip over the root tip.
Cover the slide with a paper towel and with your
thumb (or a pencil eraser), firmly press on the
cover slip; Do not twist the cover slip. The
pressure will spread the cells into a single layer.
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8. Then using the fingernail polish, carefully
paint the edges of the cover slip. Make sure
not to move the cover slip. Finally allow the
slide to dry.
9. Now you are ready to view you slide under
low power. Place your prepared slide on the
microscope.
10. Use the low power objective on your
microscope to look for thin layers of cells and
then use the 40X power objective to observe
mitotic stages in individual cells.