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Mitosis in the root tips of garlic and onion

Giorgio Carboni, April 2010
Translated by Sarah Pogue

 

CONTENTS

INTRODUCTION
PROCEDURE
CONCLUSION
 

 


INTRODUCTION

Mitosis is a process of cell replication necessary for the growth of the organism and for the substitution of "aged" cells. Upon conclusion of this process, from the original cell two cells are derived, each of which possesses the same genetic material. Above all during the growth of organisms, the cells must multiply and to do this they undergo a series of events called the cell cycle. The cell cycle has two important stages: interphase and mitosis. During interphase, the cell grows in size, doubling its DNA and preparing itself for mitosis. Mitosis involves four phases described in figures 2, 3, 4, 5 and 6. In the superior plants, mitosis occurs above all in the so-called meristem tissues. These growth tissues are found principally in the roots, in the shoots and in the cambium. There is also another process of cell division which produces the male and female germ cells. It is called meiosis and features some important differences when compared to mitosis but is not dealt with in this article.


PROCEDURE

SUMMARY
The aim of this experience is to observe and possibly photograph the process of cell multiplication called mitosis. In order to do this, we will use apical meristems of garlic or onion roots, where the growth is greatest and therefore the number of duplicating cells is relatively high. To clearly observe the different phases of mitosis, the root tissues must be well fragmented, otherwise each cell will remain attached to the others obstructing all observations. To separate the tissues into small fragments, we will need hydrochloric acid which can weaken and even remove the bonds between the cells. Coloration with Toluidine blue follows, a dye for microscopy that has a strong affinity for chromosomes. This experiment uses materials that are easily found. You can procure Toluidine blue in a shop selling products for chemical laboratories or via the internet.

MATERIALS
- a clove of garlic or an onion;
- 4 100 cc beakers or glass jars at least one of which has thin walls (e.g. yoghurt pots);
- saucepan;
- thermometer which reads at least as far as 100C;
- muriatic acid at 10% HCl;
- distilled water;
- some clean microscope slides and coverslips;
- pipette with a teat or a small bottle with a dropper;
- scissors;
- tweezers;
- razor blade;
- 2 needles or pins;
- paper towels;
- stereoscopic microscope (optional but very useful);
- biological microscope;
- camera and attachment to mount it on the microscope;
- 0.5% Toluidine blue or another nuclear dye;

PROCEDURE
- place the garlic or onion to root in a glass jar (1);
- fill the jar with tap water until the root area is covered;
- after two or three days the roots should be sufficiently long;
- cut approximately 5mm off the tips of a couple of roots (2);
- put them in a small beaker or glass jar with thin walls (3);
- place hydrochloric acid with a concentration of 1M in the beaker to a depth of about 5 mm (4);
- put the beaker in a saucepan containing a couple of cm of water at 60C and keep it in a bain-marie for approximately 6-7 minutes, or at room temperature for approximately 20 minutes;
- remove the beaker from the saucepan and transfer the root tips to a microscope slide;
- with a pipette and some distilled water, rinse away the acid;
- dry the root tips with a paper towel without touching them;
- repeat the rinsing several times;
- with a blade, shorten the root tips to 2 mm in length, keep the tips and throw away the rest (5);
- with two needles or pins and under the stereoscopic microscope, carefully chop the tips and separate the fragments;
- the root tips should come undone easily, otherwise repeat the acid treatment;
- colour the tissues with 0.5% Toluidine blue for 2 minutes;
- mount a coverslip;
- with a pipette, place a couple of drops of distilled water one one side of the coverslip and absorb the coloured water from the other so to remove the dye;
- with the biological microscope search for cells undergoing mitosis.

NOTE:
1 In principle, all of the liliaceae are suitable for this experiment, avoiding those which are poisonous. For convenience, we will use garlic and onion because they are found in almost every household. The products used must be quite fresh. To the touch they must feel quite hard. They must not have roots, and if there are any cut them away. To prevent the clove of garlic falling into the jar, insert three cocktail sticks into it at an angle of 120.
2 choose the longest roots, where the process of mitosis should be more active.
3 a glass yogurt jar is also suitable, provided that its walls are thin. Given the thickness of its sides, a drinking glass is not suitable.
4 the purpose of the hydrochloric acid is to destroy the substances that unite the cells (usually pectin), but it does not destroy the cell walls. The hydrochloric acid also has the ability to kill the cells and halt the process of mitosis. The muriatic acid that is sold in shops and supermarkets is a watery solution of hydrochloric acid which should have a concentration of about 10%. Read the packaging to verify this. If the concentration is 10%, to obtain a concentration of 1M add one part muriatic acid to two parts water (preferably distilled). Do not buy hydrochloric acid in high concentrations because it is dangerous. Get help from an adult.
5 the tissues where cell duplication is most active are those near the tip of the root.

Photographs of garlic cells in different stages of mitosis follow. All were taken with:
- an Optech mod. Biostar B5 Microscope;
- bright field illumination;
- a Lomo apo 65 X objective immersed in water;
- the photographs were taken with a Canon A630 camera, mounted on a long eye relief eyepiece made from two 8x30 binocular objectives;
- the photos, initially 3264x2448 pixels in size, were reduced to 800x600 pixels. They were then cut down to 400x300 pixels;
- the initial field was 0.142 mm on the largest side. After cutting down, this was equal to 0.071 mm;
- the difference in the colours of the photos depends on automatic or manual adjustment of the colour and contrast;
- the images refer to garlic except when otherwise indicated. At any rate, garlic and onion are both liliaceae and their mitotic processes are very similar.

 


Figure 2 - Prophase: the chromosomes begin to condense, while
around the nucleus spindle fibres develop which organise the
separation of the chromosomes into two new nuclei. The
spindle fibres are fixed to a central zone of the chromosomes
called a centromere.
Figure 3 - Prophase (as in Figure 2).

 

Figure 4 - Metaphase: the chromosomes line
up along the equatorial plane of the cell.
Figure 5 - Anaphase: the chromosome pairs divide and
the two groups migrate to opposite poles of the cell.

 


Figure 6 - Telophase: around each group of chromosomes a
nuclear membrane forms, the chromosomes disperse and can
no longer be distinguished. The spindle fibres dissolve. In the
equatorial zone of the cell a new cell wall forms and the two
cells separate. At the end, they can no longer be distinguished
from the other cells in interphase that surround them.

Figure 7 Elongated cells and nuclei of the root.
Photo reduced in size but not cut down.

 

 

CONCLUSION

There are many different versions of this experience. I chose a simple procedure that can be carried out using materials that are easily found in the home. The exception is Toluidine blue which however can be bought in shops that sell materials for chemical and biological laboratories. Almost all microscopists have this dye or another nuclear colorant. Colouration of the mitotic spindle would also be useful to more fully illustrate the phenomenon of cellular duplication. However, another colorant would be necessary, not blue but instead red or orange. This simple experiment can help give an idea of the complexity, of the precision and of the fascination of the processes that sustain the life of living organisms.

This procedure of maceration that permits the disassociation of the cells of the vegetable tissues, can also be tried for other reasons. For example, to separate the cells of leaves to observe their internal organelles, such as the chloroplasts. Unfortunately, the acid tends to alter the colour of the chloroplasts. For every tissue and for every species different procedures must be tried, including curettage and fragmentation. These methods are rather destructive, but it is often possible to find elements which have remained intact. Furthermore, if you avoid using acid, the fragments maintain their colours better.

 


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