BIOL0129 Mitosis and the Cell Cycle Cell division is the process by which eukaryotic cells reproduce. The hallmark of the process is the apportionment of
an identical genome to each daughter cell. Division of the nuclear contents is referred to as karyokinesis or, more
commonly, mitosis. Nuclear division is usually followed by cytoplasmic division or cytokinesis, the apportionment
of the cytosol and cytoplasmic organelles to the daughter cells as cell boundaries are formed. In this project, you will
study mitosis in prepared slides of the broad bean (Vicia faba) root tips. It is a convenient and very popular system
for studying mitosis because the chromosomes are very large, the diploid number is relatively low (2n = 12).
The Cell Cycle
In the life cycle of a eukaryotic cell, mitosis is part of a larger program, the cell cycle. Actively dividing cells, such
as in meristematic tissues of plants or in embryonic tissues, are alternately in mitosis and in interphase. It is during
interphase that cell growth and DNA occur, the latter during the S period. There are two other periods in interphase,
G1 (1st gap), which precedes DNA synthesis, and G2 (2nd gap), which follows it. A cell in G1 is considered to have
the 2C level of DNA. During the S period, the amount of DNA doubles to the 4C level and remains there until the
end of division. After cytokinesis, each daughter cell ends up with the 2C level of DNA. A mature eukaryotic cell
that is destined not to undergo further division is usually arrested prior to DNA syntheses, G1. There is considerable
variation both in the relative and actual duration of each stage depending on the organism, cell type, and
environmental conditions. In meristematic cells of V. faba root tips, the cell cycle lasts about 15 hours. The specific
durations of G1, S, G2, and mitosis have been determined from an autoradiographic analysis.
Mitotic Stages
In the interphase nucleus, the chromosomal material is dispersed as a rather homogeneous mass of fibers, referred to
as chromatin. There may be several clumps of condensed, deeply stained heterochromatin, and there is always at
least one nucleolus. In your preparations, nucleoli will appear as large, lightly stained, round or oval structures
within the nucleus. The stages of mitosis are prophase, metaphase, anaphase, and telophase. While each mitotic
stage is distinct, one stage does flow into the next so that the adjectives early and late are often appropriate.
Prophase:
Prophase begins when the individual chromosomes can first be discerned. Though the chromosomes are long and
thin, the double nature of each is apparent. The two subunits, termed chromatids, are joined at a narrow
chromosomal region known as the centromere or primary constriction. Throughout prophase, the chromosomes
condense and shorten. The spindle assembles in late prophase, and the chromosomes migrate toward a median
position between the poles, a process termed congression. These movements are effected by spindle fibers extending
from the poles to the chromosomes. The spindle fibers, which are composed of microtubules, terminate within the
kinetochore, a plate like structure at the centromeric region of each chromatid. Other spindle fibers extend from pole
to pole. The other event that occurs at late prophase is the disassembly of the nucleoli and nuclear envelope.
Metaphase:
At metaphase, the fully condensed chromosomes are lined up on the metaphase plate, a plane that lies midway
between the poles. Although all centromeres lie on the metaphase plate, the chromosome arms are still quite long
and extend laterally to either side of the plate. Each metaphase chromosome has a characteristic morphology
determined by its size and centromere location. Metacentric chromosomes have a median centromere and two arms
of equal length. In acrocentrics, the centromere is very close to one end, while in telocentrics it is at the tip. Another
morphological feature apparent in certain chromosomes is the secondary constriction. The secondary constriction is
1 the very thin, lightly stained chromosomal region where a nucleolus forms at telophase. The chromosomal region
distal to the secondary constriction is termed a satellite.
Anaphase:
Anaphase is the climax of mitosis. The two chromatids part at the centromere and move to opposite poles, and, in
addition, the poles move farther apart. The mechanism of anaphase movement is not fully understood but may
involve spindle microtubules sliding past each other. The shape of the anaphase chromosome depends on the
location of the centromere. The centromere leads the way to the pole so that a metacentric chromosome is V shaped,
a submetacentric J shaped, and a telocentric rod shaped. At late anaphase, the first sign of cytokinesis occurs with
the formation of the phragmoplast. The phragmoplast is a collection of dense material in small Golgi vesicles
associated with spindle fibers at the equatorial region.
Telophase:
Telophase commences when the two daughter sets of chromosomes reach the poles. Each daughter cell receives the
same number of chromosomes as had been present in the parent cell, though each chromosome now has only one
subunit. There is a general reversal of events that characterize prophase. The telophase chromosomes decondense
and become "fuzzy," the spindle fibers disassemble, and the nucleoli and nuclear envelope reassemble, the latter
from membranes of the endoplasmic reticulum. Cytokinesis proceeds with the accumulation and fusion of vesicles
at the equatorial region. The fused membranes of the vesicles produce continuous plasma membranes for the
adjacent daughter cells, and the contents of the vesicles form the cell plate, which eventually develops into the
primary cell wall.
Mitotic Index
In any population of mitotically active cells, only some of the cells are in mitosis at any one time. The percentage of
dividing cells is defined as the mitotic index. The approximate duration of mitosis can be obtained simply by
multiplying the mitotic index by the total duration of the cell cycle. You will determine both the mitotic index and
the duration of mitosis in broad been root tips in two different areas of the tip. You will then be able to compare the
two indices to determine which area of the tip is most mitotically active.
Root Tip Anatomy
2 Procedures
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With your partner, determine a testable hypothesis about which area of the root tip will contain the most
mitotic cells. The root tip can be divided into four major regions: the root cap, meristematic region,
elongation region, and maturation region. Choose 2 regions to compare, and hypothesize which one will
have the highest mitotic index.
Clean a prepared root tip slide with 70% ethanol. Although you are working in partners, each individual
will have his/her own microscope and slide.
Examine the slide with the low power (10X) and high dry (40X) objectives. Identify and draw a cell at
interphase, prophase, metaphase, anaphase, and telophase.
Examine several interphase nuclei, noting the prominent nucleoli. How many nucleoli are there per
nucleus?
Count 50 cells per region (100 per root tip) and determine the number of cells in the phases of mitosis and
interphase for each region in the three root tips. You will have counted 3 root tips and 300 cells total.
Total the number of cells in mitosis and interphase for each root tip region. Calculate the mitotic index,
and share this data with your partner (you will have 12 data points now; 6 for each region).
From the value of the mitotic index and the known duration of the cell cycle (15 hr), estimate the duration
of mitosis for each root tip region. This data should be included in a table in your lab report.
Take the average mitotic duration of each root tip region and make a graph. This graph should clearly
show any differences in duration that you observed.
Determine the relative frequency of each mitotic stage in each region. A combined figure should be used
for anaphase and telophase, since late anaphase and early telophase are difficult to distinguish. Calculate
the durations of these stages. Make two pie charts (one for each region) showing the duration of each
phase in each region.
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