SECTION -A

EXP:1 PREPARATION OFTEMPORARY MOUNT TO OBSERVE POLLEN

GERMINTAION

AIM:
To study and calculate the percentage of pollen germination on a slide.

Pollen Grain Germination: In nature, pollen grains germinate on the compatible stigma of the carpel.
This process is called pollen-pistil interaction. It is known to be an essential step in fertilisation of
angiosperms which determines the compatibility and incompatibility of both pollen and pistil. This
dynamic process involves pollen recognition followed by inhibition or promotion of pollen
germination. The pollen grains can be induced to germinate in a synthetic medium using chemicals
which favour pollen germination such as 10% sucrose solution that facilitates rapid growth of pollen
tubes. During germination, intine (inner wall) of pollen grain emerges out as pollen tube through one
of the germ pores in exine (outer wall) and the pollen tube grows into the genetically compatible
stigma only.
Out of two male gametes carried in the pollen tube, one fuses with the egg cell while, the other ruses
with the secondary nucleus. This process is known as double fertilisation, which is a characteristic
feature of all angiosperms. The pollen of different flowers might differ in their germination time and
length of the pollen tubes. They also show different sculpturing on their pollen wall.
EXINE NTINE TUBE OR TUBE
VEGETATIVE ÇELL NUCLEUS
CYTOPLAASM
CALLOSE
WALL

GERM
PORE NUCLEUS GERM PORE
A B GENERATIVE CELL
VACUOLE

MALE GAMETES

POLLEN TUBE TLHRE

NUCLEUS

REQUIREMENTS:
Mature pollens of any seasonal flower like Trade scantia, jasmine, lily, etc., cavity glass slides,
cover slips, microscope, distilled water, beaker, weighing machine, filter paper, glass rod, boric acid,
sucrose, calcium nitrate, brushes and dropper.
PROCEDURE:
Firstly, prepare a pollen germination medium by dissolving 10 g sucrose,30 mg calcium nitrate
and 10 mg boric acid in 100 mL of distilled water. Alternatively, 10% sucrose solution can also
be used as the medium.
Put a drop of this prepared medium or 109% sucrose solution with the help of a dropper in the
cavity slide.
iii. Dust the pollen on the medium on same slide with the help of a drawing brush.
Now, cover pollens with the cover slip.
 Observe the slide containing pollen under the microscope after 10-15 minutes:
vi. Finally, count the total number of nor germinating pollen and germinating pollen grains

OBSERVATION:
Count the germinated and total no. of pollens per microscopic field and tabulate your readings in the
following table
% of pollen
Name of the No. of pollens showing Total no. of germination
S.No
Flower germination (a) pollens seen (b) d 00

1 Hibiscu 10 20
10
g00 = 50%
20
CALCULATION:
Percentage of pollen germination=_00 or 100a
b
where, a = Number of pollens showing germination, b = Total number of pollens

RESULT:
All the pollens of different species in the medium germinate with different percentage. Also, there is
great variation in time taken for the germination of pollen of each species.

PRECAUTIONS:
The nutrient solution should be made carefully otherwise, pollen grains might fail to germinate.
Only few pollen grains should be dusted in the nutrient solution in order to avoid the
overloading or overcrowding.
While placing cover slip, air bubbles should be avoided else they might interfere with
observation and counting of pollens.
The entire process should be carried at room temperature.
Isolation of DNA from available plant material like Pea seed, Spinach, Onion, etc
 EXP :2 TO STUDY THE PLANT POPULATION DENSITY BY QUADRAT METHOD
AIM:
To study the plant population density by quadrat method.

PRINCIPLE:
Population Density:
It represents the numerical strength of a particular plant species in the community per unit area at a
specific time. The unit area may be small or large, depending on the size and nature of the plant
community being studied.
Population density of an organism in an ecosystem varies from place to place and depends on various
factors such as food and water availability, predation, weather conditions, birth rate, death rate and
migration rates. It can be calculated using the following formula:
Total no. of individuals of the species in all the sampling units ( S)
Density (D)=
Total no. of sampling units Studied ( Q)

Total number of individual(s) of the species in all the sampling units (S)
Total number of sampling units studied (Q)
The value thus obtained is then expressed as number of individuals per unit area.

Quadrat Method:
Quadrats are the sampling units of a known area. These sampling units must be distinct and must not
overlap each other. The quadrats having a rectangular frame and dimensions of 1m xim are most
commonly used for herbaceous plants. Quadrat method is considered as the most preferable method
to calculate population density and percentage frequency of different plant species

REQUIRMENTS:
Cotton/nylon thread (five meters), nails, a hammer or wooden quadrat of size 1mx 1m, paper, pencil,
hand lens, etc.

PROCEDURE:
i. Select an area having varied vegetation for study.
ii Fix four nails with the help of hammer in the selected area such that the area endosed by them
is a square of 1m X1m dimension. Make the boundaries of the square by tieing thread over the
nails.
Or
You can also use a commercially available ready-made quadrat of size 1m xim. Keep it over
thearea under study.
ii Note down the names of all the plant species seen in the quadrat. If the names of the plants are
not known, assign alphabets such as A, B, C, D, etc. to them.
Count the number of individuals of a plant species present in the quadrat and note down the
number in front of the species name.
Repeat the same step (4) for every plant species present in the quadrat. Record the data as
shown in the observation table ahead.
 vi Now, repeat the steps 1 to 5 to obtain data of nine more quadrats placed randomly at the site
of study. Take care that all the regions taken as sample areas should be distinct and non
overlapping.
Nal
Y
Thread

- Spedes A
-Species B

-Soocies C
Y - Specios D
W-Species E
Y - Species F

1m

OBSERVATIONS:
Write down the total number of species seen in ten quadrats. This will give an idea about the
composition of the vegetation. There will be difference in the species composition in the quadrats
made in shady areas, exposed areas with bright sunlight, dry or wet areas, etc.

Total Total
Plant
number
number of Density
Number of individuals in each quadrant individuals (D)=s/O
Species quadrants
(S
(0)
IV V VII V IX X

A 2 5 |7 10 0 3 27 0 27

1 4 8 0 3 2 20 10 2

C 3 0 2 19 10 1.9

RESULT:
The population density of a plant species in a given field can be calculated by the formula given below:
D=

PRECAUTIONS:
The measurement of the quadrats should be accurate
Count the individuals of one plant species at a time.
Squares should be marked from one field only and they should not be very far from each other.
Make sure that the vegetation is not damaged while laying the quadrat.
 EXP: 3 STUDY PLANT POPULATION FREQUENCY BY QUADRAT METHOD

AIM:
To study plant population frequency by quadrat method.
PRINCIPLE:
Population is known as the aggregation of individuals of the same kind inhabitating a particular place
or a particular geographical area at a particular time. Frequency is concerned with the degree of
uniformity of the occurrence of individuals of a species within a plant community. It is measured by
noting the presence of a species in random sample areas (quadrats) which are distributed as widely as
possible throughout the area of study. Frequency can be determined by means of sample areas in
order to check general impressions about relative values of species. Frequency is the number of
sampling units (as%) in which a particular species (A) occurs. The frequency of each species (sps. A or
sps. B or sps. X, etc) is expressed in percentage and is calculated as follows:

% Frequencv or Frequencv index (F)

Number of sampling units (quadrats) in which the species occurs (N)
Total number of sampling units (quadrats) employed for the study (Q)

REQUIREMENTS:
Cotton/nylon thread of S meters, nails, hammer, paper, pencil and magnifying lens.

PROCEDURE:
i. Select a field and lay a quadrat with the help of meter scale.
Measure area of about lmx 1m with the meter scale. Hammer the nails fimly at coners and
tie the thread on the nails.
ii Divide each quadrat into 16 small squares by tying thread at a distance of 25 cm each on either
side, fix nails to the thread to mark these small squares.
iv. List the names of the plant species seen in the quadrat (if the name is not known mark these as
species Aor B, etc. and if the same species is seen in other quadrats assign the same alphabet).
V. Similarly, lay ten more quadrats randomly in the field of study and record the number of
individuals of each species.
vi. Calculate the percentage frequency of occurrence of plants using the formula given below

Frequency Percentage for a species = No.of quadrants in which species appeared(N)v100

Total number of quadrants studied()

where, F= Frequency or frequency index

N= Number of sampling units (quadrats) in which the species occurs
Q=Total number of (quadrats) employed for the study
 Nail

-Thread
V- Speces A
A-Species 8
-SpeciesC
1m2
1m Y-Species D
Y
W-Species E
Y -species F

1m

OBSERVATIONS
Record the total number of species seen in the eleven quadrats. This will give an idea about the
composition of the vegetation. There will be a difference in the species composition in the quadrats
made in shady areas, exposed areas with bright sunlight, dry or wet areas, etc. Observe that the
frequency of occurrence is not the same for all species.
Number of Percentag
quadrats in
Plant which the Frequenc
Quadrats employed in the study
Species
species is F=
present (N) N/Q*100
V V V VI X
X P 7 70%
2 20%
10%

RESULT:
The frequency percentage of a given species F Ne100

PRECAUTIONS:
The measurement of quadrats should be accurate.
The string or cord used should not be very thick.
One individual of a species should be counted only once in the quadrat.
Make sure that the vegetation is not damaged, while laying the quadrat.
 EXP: 4 PREPARATION OF TEMPORARY MOUNT OF ONION ROOT TIPS TO STUDY
MITOSIS.
AIM:
To prepare a temporary mount of onion root tips to study mitosis.

Requirements:
Onion bulbs, wide mouthed glass bottle, corked tube, petri dishes, scissors, forceps, needles, methyl
alcohol, acetic acid, N/10 HCL, acetocarmine, distilled water, spirit lamp, compound microscope, slides,
blotting paper, cover slips etc.
vascular
cyinder Cortex

ep derrmis

zone ol
hair root differentlation

Three
Primar
meristems: zone of
Protoderm oiongation
"Ground
meristem
"Procambium

zone ot
apical division
meristem

root cap (B)

Procedure:
A Growing Onion root tips:
i. Select medium onion bulbs and carefully move remove the dry roots attached to them.
i. Place them in a bottle filled with water in such a way that only roots touch the water level.
ii. Check the water level in the bottle everyday and add few drops water periodically in order to
compensate any evaporation losses.
iv. Keep them in position for about 3-6days. New roots may take 3- 6days to grow.
V. Cut 2-3cm long freshly grown roots, 2hrs after the sun rice and keep harvested roots in fixative
(Glacial acetic acid and ethanol in 1:3 ratio) for 2hours.
vi. Transfer roots from fixative to 70% al co hol.
vil. These root tips are ideal material for the study of mitotic cell division.
 B. Preparation and study of slide
i. Take one or two preserved roots and wash them thoroughly in water on a clean slide.
i. Place one drop of N/10 HCL on the root tip followed by 2-3 drops of acetocarmine stain on it.
Leave it for 5-10minutes
ii. Slightly warm the slide on spirit lamp. Care should be taken that the stain is not dried up.
0v. Blot the excess sta in using blotting paper.
V Cut the more stained tip portion of root and retain it on slide and discard the remaining portion.
vi. After 10-20 seconds, put one or two drops of water and blot them carefully using blotting paper.
vii. Gently squash the root by tapping the cover slip with the blunt end of a needle so that
meristematic tissue of that root tip and is smashed and spread as a layer.
vii. There should no air bubbles under cover slip.
ix. Gently warm the slide over a flame and seal the margins of cover slip using paraffin wax.
X. Slide is now ready for the study of mitosis.
xi. Observe it in a compo und microscope.

Dropper
-Plane side
Glycerine

OBSERVATIONS:
Under lower magnification of the microscope, rectangular cells with pink nucleus are seen scattered.
Under higher magnification of the microscope different phases of mitosis become distinct. The stages
of mitosis can be broadly categorised into two main parts, i.e., karyokinesis followed by cytokinesis.
Those cells which are not in the phases of cell division are considered to be in the interphase. You will
observe that most cells in a microscopic field are in interphase.
Interphase

-centrioles
nucleus

duplicated
cytoplasm chromosome
nucleolus
i. It is a non-dividing phase of the cell cyde occurring betweentwo successive cell divisions.
ii The cells are mostly rectangular, oval or even circular in shape.
 iii. The nudeus is homogenous and looks granular in origin.
iv Nuclear envelope is distinct,
V. Chromatin fibres appear in the form of an interconnectedmass within the nucleus.
vi Nucleolus is well observed inside the nucdeus.

Stages of Mitosis
a) Prophase
i. Nuceus is enlarged and occupies most of the cell volume. Intact nuclear outline is seen.
ii. Chromatin network gets condensed and appears as long thread-like structures called
chromosomes.
ii. Nuclear membrane may start disappearing.
0v. Nucleoli may or may not be visible.
v. If the cell is in early stage of prophase, then the chromatin fibres are very thin. However, in the
cells at late prophase, comparatively thicker chromatin fibres would be visible it.

aster centromere

nuclear membrane spindle fibre

(a) Early prophase (b) Late prophase

b) Metaphase

Centromere
Spindle fibres

0. Chromosomes become shorter and thicker and hence become distinct and clearly visible under
the compound microscope.
ii. Nuclear membrane completely disappears.
ii. Chromosomes orient themselves towards the equator with their centromeres arranged on an
equatorial line forming metaphase plate. Out of two chromatids of each chromosome, one
chromatid faces one pole and the other chromatid faces the opposite pole.
0v. Series of spindle fibres attach the centromeres to the opposite poles.
V. Nucleolus is not observed during metaphase.
 c) Anaphase
Daughter
chromosomes

Spindle fibres

cell membrane

(a) Early Anaphase (b) Late Anaphase

The two sister chromatids of each chromoSome separate due to the splitting of centromere
and move towards the opposite poles.
ii. The daughter chromosomes (separated chromatids) appear in V, J, Land I shapes, depending
upon the position of the centromere in the chromosomes.
iii, Anaphase is designated as early, mid and late stages depending upon the position of moving
chromosomes toward the opposite poles.
d) Teloph ase

-Daughter nuclei

Chromosomes reach to the

opposite poles, lose their individuality and look like a mass of chromatin again.
Nudear membrane reappears to fom the nuclei of two future daughter cells.
Nucleolus gets recon stituted.
Two daughter nuclei are thus formed and appear to be similar to the parent nucleus both
quantitatively and qualitatively.
Cell plate
Cytokinesis
Daughter cell

(A) Animal cells (B) Plant cells

In plant cells, cell plate is formed in the middle after telophase. The plate can be seen to extend
outward to ultimately reach the margin of the cell and divide the cell into twO equal halves. Such cell

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plates are characteristic of plant cells. In animal cell, furrow appears in the membrane at both sides
and grows inward to divide cells into two.

RESULT:
All the stages of mitotic cell division are clearly visible in the slide prepared from onion root tips.

PRECAUTIONS:
Always clean the slide and cover slip thoroughly before and after use.
Care should be taken that there should be no air bubbles under the cover slip
iii The base of the onion bulb should exactly be in contact with water, while growing the roots.
iv. Always filter the acetocarmine stain before use.
V Material should be mounted on the centre of slide.
vi. Also, the material should only be warmed gently, i.e. do not allow solution to boil on slide while
warming.

EXP: 5 ISOLATION OF DNA FROM AVIALBLE PLANT MATERIAL

AIM:
To isolate DNA from available plant material like pea seed, spinach, onion, etc.

REQUIREMENTS:
Plant material (like pea-seed, spinach leaves, onion or cauliflower shavings), mortar and pestle,
beakers, test tubes, enzymes (like cellulase, protease, lipase and ribonuclease), ethanol, spool, glass
rod, etc.
PROCEDURE:
i. Take small amount of plant material (onion or cauliflower shavings) and grind it in a mortar
with the help of pestle. Alternatively, the plant material can be homogenised in a blender and
can be filtered easily through the muslin cloth.
ii. Now, pour the filtrate into a boiling test tube.
ii. Break the cell wall and envelope of plant cell by treating the material with the enzyme
cellulase.
0v. Now, the same material is treated with enzyme protease to remove histone proteins.
Now, add ribonuclease in it to dissolve the associated RNA followed by addition of lipase to
dissolve the lipids present in the sample.
vi. Gradually pour twice the volume of ice-cold 95% ethanol into the test tube. This will alow DNA
to precipitate out.
vii. The precipitated DNA can be spooled with the help of glass rod. It appears as the winding of the
fine threads of DNA.
OBSERVATIONS:
Shiny white DNA can be seen at the junction of solution and ethanol. This DNA represents all the DNA
found in plant cells. The chromosomes were broken in the process and the DNA gets precipitated due
to the chemical treatment.

RESULT:
DNA appears as white precipitate of very fine threads on the spool.

PRECAUTIONS:
" Fresh plant materials should be used for extraction of DNA.
The glass wares must be thoroughly cleaned and dried before the experiment starts.
The enzymes and chemicals used for the experiment should be of standard quality.
Always use distilled water to make the solutions.
Chemicals and solutions should be prepared carefuly in order to avoid any wastage.

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