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DATE: 2 JULY 2014

1. To determine the effect of colchicine on onion root tips.
Mitosis is the nuclear division of a eukaryotic cell which produces two daughter
cells identical to the dividing parent cell. Mitosis is important for growth in our human
body by replacing old cells and tissue repair. Mitosis also is part of asexual reproduction
for single celled and some multicellular eukaryotes. Mitosis is a one portion of the cell
cycle. One complete cell cycle is divided into interphase, mitosis and cytoplasmic
division in eukaryotic cells. Most of the time, the cell are in interphase, which during the
cell synthesizes nuclear components and cytoplasmic.
DNA replication also occurs in interphase. This process involves the precise
duplication of the entire DNA in the nucleus, in preparation for mitosis. Mitosis creates
two identical nuclei, one for each daughter cell in the physical division of the nucleus.
Cytoplasmic division results in the partitioning of the cytoplasmic components and the
physical separation of the two new daughter cells.
Mitosis has four basic stages; they are prophase, metaphase, anaphase
and telophase. Prophase is where the replicated chromosomes condense into compact
rods. Each rod consists of two sister chromatids joined at the centromere. The spindle
apparatus made of microtubules and centrioles at opposite poles of the cell. Microtubules
attached at the centromere of each chromosome and then connect the chromosomes to the
spindle apparatus. The nuclear membrane breaks up within nucleus during these changes.
In metaphase, the chromosomes are in their most condensed form, line up at the spindle
equator. For anaphase stage, the two sister chromatids break their attachment to each
other. As separate chromosomes, they are pulled to opposite spindle poles by the
microtubules. The last stage is telophase. Now, the cell has two clusters of chromosomes,
which are located at opposite poles. A new nuclear membrane begins to form around each
of these clusters. This is resulting in two new nuclei, one for each new daughter cell. The
DNA replication during interphase is to ensure that each daughter cell receives a full
chromosomal complement of 46 chromosomes. Cytoplasmic division is started at this
point in mitosis. In plants, a cell plate is formed by vesicles filled with cell wall
material. These vesicles combine at the former site of the spindle equator. Cellulose
deposits accumulate at this plate. Eventually, a new cell wall is formed and separating the
parent cell into two daughter cells.
The Role of Spindle Fibers in Mitosis
Spindle fibers play an important role in mitotic process due to them contains a
highly organized array of microtublules, which composed of globular proteins in long,
hollow, unbranched tubes. Microtubules are components of other cellular structures,
including cytoskeleton. They form the core of both cilia and flagella. Besides involving in
separating chromosomes during mitosis, microtubules are structural supports and
organizers for cells. In plant cells, it maintains cell shape through their influence on the
formation of the cell wall during interphase. Apart from that, microtubules also maintain
internal organization of cells and are responsible for intracellular motility of

macromolecules and organelles. Hence, colchicines, a microtubule disrupting drugs, do

bring a serious effect to it. The transport of materials from one membrane compartment to
another depends on the presence of microtubules because specific disruption of these
cytoskeleton elements brings the movements to a halt.
Colchicine is an alkaloid extracted from Colchicum autumnale L. which binds to
tubulin dimmers in vitro and results in the formation of a tubulin-colchicine complex
acting primarily to prevent microtubules assembly.
It is believed that the organization of the mitotic apparatus depends upon balance
between the elements of cytoplasm and the mitotic apparatus. Any chemical that disturbs
this delicate balance will prevent the formation of the mitotic apparatus, and colchicine is
said to have this property. If the formation of the spindle apparatus in living cells is
inhibited, these cells will stop dividing. If the inhibitor of the spindle apparatus formation
is eliminated, spindle apparatus will be rebuilt and cell division will resume for these
In vivo, colchicines interfere with the cytoskeleton by inducing microtubules
depolymerization and preventing the formation of the mitotic spindle. Inhibiting spindle
assembly induces the formation of polypoid cells due to the lack of normal chromatid
Polyploidy is the process of genome doubling that gives rise to organisms with
multiple sets of chromosomes. Ploidy refers to the number of complete genomes
contained in a single cell. Generally, polyploid organisms contain combination of the
chromosome sets found in the same or a closely related diploid species. Polyploidy can
arise from spontaneous somatic chromosome duplication or as a result of non-disjunction
of the homologous chromosomes during meiosis resulting in diploid gametes. When
polyploidy artificially induced by treatment with drugs, such as colchicine, it inhibits cell
division. Most somatic cells of the organism are occurring polyploidy and it can be
restricted to a specific tissue too.
Polyploidy is common among plants. In fact, it is a major source of speciation in
the angiosperms. There is evolution by polyploidy. As polyploidy is the addition of one or
more complete sets of chromosomes to the original set, if there are two copies of each
autosome, it is called diploid. If four copies of each autosome, it is called tetraploid. If six
copies of each autosome, it is called hexaploid. The gametes of diploids are haploid,
those of tertraploids are diploid, and those of hexaploid are triploid, and so on.
There are two main types of polyploidy: autopolyploidy and allopolyploid.
Autopolyploidy, which is genome doubling, is the multiplication of one basic set of
chromosomes. Meanwhile, allopolyploidy is the combination of genetically distinct, but
similar chromosome sets. Autopolyploids are derived from within a single species;
allopolyploids arise via hybridization between two species. Autopolyploidy may be

common in plants, although its prevalence may be underestimated in the taxonomic

literature. One species that is doubtlessly a true autopolyploid, rather than an
allopolyploid derived from two very similar diploids, is the potato, Solanum tuberosum.
Allopolyploidy is much more common in nature than autopolyploidy. About 80% of all
land plants may be allopolyploids.

Squash Preparation of Untreated Onion Root Tip.

2 to 3 root tips were placed on a clean watch glass.

9 drops of 1% aceto-orcein and1 drop of 1M HCl were added.

Watch glass was warmed by over a spirit lamp until white vapors were seen (~1 min)

The watch glass was closed with another watch glass and allowed it to cool for about 5 to
10 mins.

The tip of the root (~ 1min) was cut and discarded the rest.

1 or 2 drops of 1% aceto orcein were placed on the glass slide with the tip of root.

The glass rod was used to tap gently on the root tips so that the cells get separated.

The squash was covered with cover slip.

The excess stain was blotted by tissue and without removing the coverslip.

The slide was heated gently over a spirit lamp.

The slide was inverted carefully onto a filter paper and pressed gently with thumb
without shifting the slide.

The slide was observed under the microscopic.


Squash Preparation of the Colchicine Treated Onion Root Tip.

Make a squash preparation with the treated root tips following the same
method as for untreated root tips.

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