SCHOOL OF INTERNATIONAL TROPICAL FORESTRY

FA20203
GENETICS AND PLANT BREEDING

LABORATORY 1
Part 1: Mitosis in Plant Root Tips

Introduction

Mitosis is the nuclear division associated with the asexual division of cells. In multicellular diploid
organisms, mitosis takes place during the division of somatic cells. Mitosis distributes
chromosomes equally to each of daughter cells ensuring that every cell in an organism carries
the same set of chromosomes. Mitotic activity is the foundation for the development and growth
of organism. Two main division activities of living cells are karyokinesis and cytokinesis.
Karyokinesis is the process of partitioning genetic material into daughter cells during nuclear
division, which is complex and requires great precision. The two daughter nuclei produced will
each have a chromosome composition identical to that of the parent cell. Karyokinesis is
followed by cytoplasmic division or cytokinesis. Cytoplasma material is partitioned into two
parts, and then encloses both new cells within a distinct plasma membrane. Cytoplasmic
organelles are replicated or synthesized anew in each cell.

Objectives

To prepare an acetocarmine squash of a root tip to demonstrate the process of mitosis in onion
roots

Materials

 Onion and garlic roots  Cover slips

 IM HCL  Watch glass
 45% acetic acid  Glass rod
 Aceto-carmine stain  Forceps
 Microscope  Scalpel
 Microscope slides  Alcohol lamp

Procedures

Root-tip squash preparations for mitotic chromosome analysis of regenerated plants

1. Use a clean pair of forceps to collect healthy roots into a small vial containing distilled water.
2. Transfer the roots to a suitable pre-treatment (eq. 8-hydroxyquinoline 0.29g per L – dissolve
at 60ºC) as quickly as possible and incubate for the appropriate time and temperature.
3. Fix the roots by transferring them into a fresh mixture of 3:1 absolute alcohol:glacial acetic
acid and incubate for at least 24 hours at 4ºC.
(Steps 1-3 prepared beforehand)
4. Hydrolyze the roots by incubation in 1M HCL at 60ºC for 10-15 minutes.
5. Wash the roots briefly in distilled water.
6. Place the root onto a clean glass slide. Remove the translucent root cap at the extreme tip
and place the root-tip in a small drop of 45% acetic acid. Mix in a small drop of aceto-
carmine.
7. Tap the root-tip thoroughly in the drop with a flat ended glass rod. Remove remaining large
pieces with a needle.
8. Place cover slip over the drop and heat very gently over a small flame without creating air
bubbles.
9. Squash under filter paper, vertically downwards using the thumb. Do not rock your thumb
during the squash as this will roll the cells.
10. Examine under the microscope. First under lower magnification power (20X) and gradually
under high magnification power (100X).
 SCHOOL OF INTERNATIONAL TROPICAL FORESTRY

FA20203
GENETICS AND PLANT BREEDING

Observation

Observe the different cells and determine the various stages of mitosis. Illustrate and explain the
various mitotic stages in your laboratory report. If you can observe chromosome morphology,
identify and explain them as well.

Questions

a. Can you locate the various stages of mitosis – prophase, metaphase, anaphase and
telophase?
b. Can you discern anything of chromosome morphology? For example that some of the
chromosomes are composed of two chromatids or the location of the centromere on any such
chromosome?
c. Can you determine the number of chromosomes in any cells?
d. What mitotic states are most prevalent?
e. Are any mitotic stages completely lacking? Why?
f. Can you identify and explain the following occurrence in the cells:

i) Synapsis
ii) Bivalent
iii) Chiasmata
iv) Crossing over

Part 2: Meiosis

Introduction

In general, meiosis occurs either in plants or in animal at the time of sex cell (gamete) formation.
In angiosperms or flowering plants, meiosis occurs at two locations in the flower. Pollen mother
cells (PMC) located in the anthers undergoes meiosis and form microspores. The haploid
nucleus of each microspore subsequently divides mitotically to give rise to a three-nucleate male
gametophyte. Two of the three nuclei are sperm nuclei that function in a double fertilization
process. Meiosis also occurs in a sporangium called the ovule. This ovule is located inside the
ovary of the flower pistil. In ovule, meiosis leads to the formation of a megaspore. The
megaspores divide mitotically to form a female gametophyte, one nucleus of which is an egg.

Objectives

1. to prepare an acetocarmine squash of anthers from a flower bud to demonstrate the stages of
meiosis in microsporogenesis
2. to identify the basic features of meiosis in plant gametogenesis

Materials

 Prepared slides of microsporogenesis in  Cover slips

Lily plant  Watch glasses
 Lilium  Scalpel
 Acetocarmine stain  Forceps
 Compound microscope  Alcohol lamp
 Stereo dissecting microscope
 Microscope slides
 45% acetic acid
 SCHOOL OF INTERNATIONAL TROPICAL FORESTRY

FA20203
GENETICS AND PLANT BREEDING

Procedures

1. Dissect out a single anther form bud and place on a clean slide.
2. Either smear the anther on the slide with scalpel or needle-knife and add a drop of acetocarmine or
break up the anther in a drop of stain.
3. Carefully remove all debris and large pieces of material leaving the contents of the anther as a cell
suspension in the drop of stain.
4. Cover with a cover glass, heat gently in an alcohol lamp and then squash immediately between filter
paper with hard pressure.
5. Examine the slide under microscope as in the mitosis procedures. Carefully study the cells in the
various meiotic stages.

Questions

i) Can you locate the various stages of Lily meiosis? State and explain.
ii) What meiotic stages are most prevalent?
iii) Can you determine the number of chromosome in PMCs and the number of chromosomes in the
microspores?
iv) Can you see chiasmata at diplonema and diakinesis?
v) How many chiasmata occur per bivalent (tetrad)?
vi) How many tetrads can you count at diakinesis?
vii) How many chromosomes are present in a PMC nucleus at diakinesis? Are the cells haploid or
diploid?
viii) How many chromosomes will be present in each nucleus at telophase I and at prophase II in the
plants given?
ix) Can you differentiate between cells in metaphase II and cells in metaphase I?
x) At telophase II in the process of microsporogenesis, a quartet of cells is formed. What name is given
to each of there cells? What changes do these cells subsequently undergo?

References

Klug, W.S., M.R. Cummings, C.A. Spencer, M.A. Palladino. 2010. (7 th edition). Essentials of Genetics.
Pearson Education, Inc.