Mitosis and the Cell Cycle in Onion Root-Tip Cells

 

Introduction

Cell division : The genetic information of plants, animals and other eukaryotic organisms resides in several (or many) individual DNA molecules, or chromosomes. For example, each human cell possesses 46

chromosomes, while each cell of an onion possesses 8 chromosomes. All cells must replicate their DNA when dividing. During DNA replication, the two strands of the DNA double helix separate, and for each original strand a new complementary strand is produced, yielding two identical DNA molecules.

DNA replication yields an identical pair of DNA molecules (called sister chromatids) attached at a region called the centromere. DNA replication in eukaryotes is followed by the process called mitosis which assures that each daughter cell receives one copy of each of the replicated chromosomes. During the process of mitosis, the chromosomes pass through several stages known as prophase, metaphase, anaphase and telophase. The actual division of the cytoplasm is called cytokinesis and occurs during telophase. During each of the preceding stages, particular events occur that contribute to the orderly distribution of the replicated chromosomes prior to cytokinesis.

The stages of mitosis

·       Prophase: During prophase, the chromosomes supercoil and the fibers of the spindle apparatus begin to form between centrosomes located at the pole of the cells. The nuclear membrane also disintegrates at this time, freeing the chromosomes into the surrounding cytoplasm.

·       Prometaphase. During prometaphase, some of the fibers attach to the centromere of each pair of sister chromatids and they begin to move toward the center of the cell.

·          Metaphase. At metaphase the chromosomes have come to rest along the center plane of the cell.

·        Anaphase. During anaphase, the centromeres split and the sister chromatids begin to migrate toward the opposite poles of the cell.

·     Telophase. During telophase, the chromosomes at either end of the cell cluster begin to cluster together, which facilitates the formation of a new nuclear membrane. This also is when cytokinesis occurs, leading to two separate cells. One way to identify that telophase has begun is by looking for the formation of the cell plate, the new cell wall forming between the two cells.

Viewing mitosis in onion root tips.

Why use onion roots for viewing mitosis?

·       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.

·       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.

·       The chromosomes can be stained to make them more easily observable.

 

Regions of Onion Root tips:

There are three cellular regions near the tip of an onion root.

·       The root cap contains cells that cover and protect the underlying growth region as the root pushed through the soil.

·       The region of cell division (or meristem) is where cells are actively dividing but not increasing significantly in size.

·       In the region of cell elongation, cell are increasing in size, but not dividing.

 

Requirement:

Sample:                      Onion root (Red onion)

Chemical :                  0.5% toluidine blue, 1-M HCl

Instrument :               Compound light microscope,

glass ware:-               Clean glass slide,coverslips,

labware:-                    Distilled water ,Slide holder/clothespin, Razor blades, dropper bottle,                                                  Paper towels

 

Procedure:

·       Obtain an onion bulb that is just beginning to show the emergence of roots. Cut off the first 1 or 2 mm of the root tip and place it on a clean glass slide.

·       Cover the root tip with two or three drops of 1 M-HCl. Using forceps or a clothespin to hold the slide, pass the slide over the flame of a Bunsen burner for 2 - 3 seconds. (Remember to wear your safety glasses.) Be careful not to get HCl on your hands.

·       Using the edge of a paper towel, blot around the root to remove excess HCl and cover the root tip with 0.5% aqueous toluidine blue (less than a drop) Again quickly pass the slide above the Bunsen burner flame four times without boiling. Let stand for one minute

Prepare the root tip squash.

1.      Transfer a root to the center of a clean microscope slide and add a drop of water. Using a razor blade cut off most of the unstained part of the root, and discards it.

2.      Cover the root tip with a cover slip, and then carefully push down on the cover slide with the wooden end of a dissecting probe. Push hard, but do not twist or push the cover slide sideways. The root tip should spread out to a diameter about 0.5 – 1 cm.

3.     Using your microscope, locate the meristematic region of the root tip. Examine the slide at high power and identify chromosomes at the various stages of mitosis.

 

Result

 

Onioan Root tip squash

 

Find and Draw a cell showing each stage of mitosis