Experiment 9: Study of Meiosis in Onion Bud Cells or Grasshopper Testis Using Permanent Slides

AIM

To study the meiosis in onion bud or grasshopper testis using permanent slides.

PRINCIPLE

Meiosis is reduction division that reduces chromosome number by half and produces four haploid gametes from a diploid parent cell. It consists of two successive divisions, Meiosis-I and Meiosis-II, with single DNA replication.

Meiosis-I is reductional division where homologous chromosomes pair, exchange segments by crossing over and separate. Prophase-I is long and divided into leptotene, zygotene, pachytene, diplotene and diakinesis stages.

Meiosis-II is similar to mitosis without DNA duplication. Sister chromatids separate in anaphase-II, forming four haploid nuclei. Grasshopper testis and onion floral buds show active meiotic divisions and are ideal materials.

REQUIREMENTS

• Permanent slides of all the stages of meiotic division
• Compound microscope

PROCEDURE

1. Focus the slide under low power (10x) of a compound microscope.
2. Observe the dividing cells carefully.
3. Look for the nucleus, chromosome, etc. in each cell.
4. Use pointer in the objective lens to point out a specific stage of meiotic cell division to be seen in high power.
5. Observe the stage of the cell pointed by pointer under high power (45x).

OBSERVATION

Different stages of meiosis have different special features. These features are summarised with proper labelled diagram as given below:

MEIOSIS-I

A. Prophase-I

It is a complex phase characterised by number of events. It is divided into five main sub-stages:

a) Leptotene

• Nuclear membrane and nucleolus are not clearly visible. Fine network of thin chromatin threads is seen.
• These are chromatin fibres in condensed form called chromosomes.

b) Zygotene

• Each pair has two chromosomes similar in their length and their centromere position.
• Chromosomes become more distinct as they become much shorter than before.
• Pairing of homologous chromosomes takes place and is called synapsis.

c) Pachytene

• The homologous pairs are clearly visible.
• Each chromosome has two chromatids and thus each bivalent consists of 4 chromatids. Hence, the chromosomes exhibit tetrad configuration.
• Crossing over, i.e., exchange of chromatid segments takes place between non-sister chromatids of homologous chromosomes.

Diplotene

• Diplos means double, so each homologous chromosome has two chromatids that show distinct separation from each other except at some points.
• The attachment points of two homologous chromosomes are called chiasmata.
• These chiasmata represent the site of crossing over.
• Nuclear membrane is disappearing and nucleolus is disappearing.

d) Diakinesis

• Bivalents condense further during this stage and appear to be more shortened, thick and prominent than before.
• Chiasmata are clearly visible.
• All homologous pairs appear in a scattered form within the cell.
• Nuclear membrane and nucleolus have disappeared completely.
• Spindle formation can be seen in its early stages.

B. Metaphase-I

• The homologous chromosomes are still in pairs and are arranged along the equatorial plane of the cell.
• At this stage, number of bivalents can be counted.
• Chiasmata may still be seen in few bivalents.
• The spindle fibres attach themselves to the centrosome of each chromosome pair.
• Chromosomes are arranged along the equatorial plane of the spindle fibre.

C. Anaphase-I

• As a result of shortening of spindle fibres, the paired chromosomes start separating.
• At the end of anaphase-I, the chromosomes assemble at two poles.
• This results into the reduction of chromosomes number to half.
• Each chromosome has two chromatids at this stage.
• Separation of homologous chromosomes is observed.

D. Telophase-I

• Chromosomes present at the two poles appear decondensed.
• The nuclear membrane is formed around the two new daughter nuclei.
• Nucleolus also reappears.
• Thus, each nucleus formed has half number of chromosomes as compared to the nucleus of the parent cell.
• Cytokinesis follows telophase-I forming two daughter cells.

MEIOSIS-II

Meiosis-II is similar to mitosis without duplication of DNA. It is divided into following stages:

A. Prophase-II

• The chromosomes reappear as distinct rod-shaped or thread-like chromatin fibres.
• Each chromosome has two chromatids.
• Nuclear membrane and nucleolus start disappearing.
• The chromosomes become short by coiling and condensation.
• A new spindle forms around the chromosomes.

B. Metaphase-II

• This phase is similar to that of mitotic division.
• The chromosomes having two chromatids attached at the centromere are observed arranged at the equatorial plane of the cell.
• Metaphase-II chromosomes line up at the equator.

C. Anaphase-II

• The centromere of each chromosome divides into two so that each chromatid gets its centromere.
• Shortening of the spindle fibres occurs so that chromatids are pulled apart towards their respective poles.
• The two chromatids of each chromosome after separation appear to lie at the two poles of the cell.
• Centromeres divide, chromatids move to the opposite poles of the cells.

D. Telophase-II

• The chromatids (now chromosome) on their respective poles, now uncoil and form the chromatin network again.
• Nuclear membrane and nucleolus are reformed.
• Four haploid nuclei are seen in each cell (male or female gamete).
• A nuclear envelope forms around each set of chromosomes and the cytoplasm divides.

RESULT

The slides under observation revealed all the characteristic features of the meiotic cell division occurring in the onion bud or grasshopper testis.

CONCLUSION

Meiosis consists of reductional Meiosis-I and equational Meiosis-II producing four haploid cells from one diploid cell. Crossing over and chiasmata formation during prophase-I introduces genetic variation, while reduction in chromosome number maintains species chromosome constancy.

PRECAUTIONS

• Handle the permanent slides cautiously so that they do not break.
• Focus each slide first under 10x magnification of light microscope and then under 40x magnification to get the better view of dividing cells.