PART

II

CORE EXPERIMENTS
 SECTION A
Study/observation of the following experiments :
1. Study of pollen germination on a slide.
2. Study of plant population density by quadrat method.
3. Study of plant population frequency by quadrat method.
4. Study of mitosis in onion root tip (preparation).
5. Isolation of DNA from available plant material such as spinach leaves, green pea seeds,
papaya etc.
CORE
PER Study of Pollen
Germination
1
INTRODUCTION
1 Pollen grain or microspore is the first cell of male gametophyte.
2. The development of male gametophyte is precocious, i.e., it begins inside the micro- sporangium
or pollen sac.
liberation it becomes
3. The pollen grain is uninucleate in the beginning but at the time of
2celled-a small generative cell and alarge tube or vegetative cell.
the stigmatic secretion
4 On the stigma, the pollen grain absorbs water and nutrients from
through its germ pores.
into the pollen tube
5. The tube cell gives rise to a pollen tube. The generative cell also descends
and divides into two male gametes.

EXPERIMENT 1.1
 Comprekenstue Laboratory
22
Exine
Manualin Biology
Intine
Germ
pores

Pollen.
tube

Tube nucleus

Male gametes

Fig. 1.1. Germination of pollen grains.

PRECAUTIONS
1. Flowers should be freshly plucked.
2. Use clean slide to
observe the pollen grains.

VIVA VOCE
Q.1. What is the shape of a
pollengrain?
Ans. Itis commonly globular in
Q. 2. outline, though several other shapes are also
What is palynology ? found.
Ans. The study of pollen grains is
Q.3. What is the called palynology.
Ans. It is made up composition
of wall of pollen
of two
layers, outer exine and inner
grain?
Q.4. What is the intine.
Ans. Intine is
chemical nature of the two layers of the wall of pollen
pecto-cellulosic
called sporopollenin.
in nature and
exine is made of highly grain ?
Q, 5. What is resistant fatty substance
tectum ?
Ans. It is the
discontinuous surface layer of the exine of the
characteristic
Q,6. What is the sculpturing or designs over the surface of pollen grain wall, which provid
Ans. It an help a importance of tectum to a pollen grain.
species. taxonomist toidentify the pollentaxonomist ?
grains and refer them to their family, genus
 Study of Plant
M2ENT Population
Density
INTRODUCTION
() Population is defined as anearly permanent aggregation of individuals of the same kind
which inhibit aparticular space or geographical area at aparticular time. It is subordinate
toa species as a unit of cooperative aggregation of individuals.
() The number of individuals in apopulation never remain constant. It may increase or decrease
due to many factors like birth rate, death rate, migration etc.
(i) The number of individuals of a species present per unit area or space of a given time 1s called
population density.
(v) Population density is calculated by counting all the individuals present at a given time in a
given space or area divided by the number of units of area or space.
D=
S
Here, D =population density, N = number of individuals and S = units of space.
() The population density of different plant species can be determined by laying quadrats/ land
segments of suitable size and recording the number of individuals of each species occurring
in the quadrat.

EXPERIMENT 2.1
AIM: To study population density of different lant species of agiven area.
REQUIREMENTS
Metre scale, string or cord, nails, paper, pencil etc.
PROCEDURE
(a) Determination of the size of Quadrat. Prepare a Lshaped structure in the fheld of 1 m
x1 m by using 3 nails and tying a string with them. Now measure 10 cm on one side of
the arm of Land then the other. Prepare 10 x 10sq. cm area using another piece of
string.
Count the number of species occurring in this area. Increase this area to 20 x 20 sq. cm and
similarly record additional species occurring in this area. Repeat the same procedure till
lx1 sq. marea covered. Record the observations as follows:
 24 Comprekenswe LaboratoryhManual in E
1m

90
Biologyx
80

70

60

50

40

30

20

10

0 10 20 30 40 50 60 70 80 90 1m
Fig. 2.1. Procedure to find out
minimum size of the quadrat.
Table 2.1. Total number of
species and the area
S
No. Area

1. 10 x10 sq. cm
Total no. of species
2. 20 x 20 sq. cm
3. 30 x30 sq. cm
up to
100 x 100 sq. cm

Using the above

size of the quadrats on Xrecorded data, prepare a graph. Number of
species are plotted on Yaxis and
increase. This point denotesaxis. At one point of graph, curve starts flattening
consideration. minimum area of the quadrant suitable for or shows only a grau
the study of an area
uu
CoreExperiments
25
8

25 50 75 100 cm
Fig. 2.2. Species area curve to determine the size of the
quadrat.
(b) Determination of Population density. Take a quadrat of suitable size, lay it randomly
at number of places. Count the number of each plant species present in the quadrat. If the
number of plants in the quadrat is large, the quadrat can be divided into smaller sub-units
and the sum total of all the sub-units will give the number of individuals of a species in the
quadrat. Calculate the population density using the following formula.
Total No. of individuals in all the quadrats studied
Population Density =
Total No. of quadrats studied
Record the data in the observation table.

50
cm

ss 50 cm
Fig. 2.3. A quadrat.
26 Comprekenslve Laboratory
Manualin
Nail Biol gy-a
Species No.1
Species No.2
50 cm
Species No.3
Species No. 4
-String
Species No. 5

Nail
50 cm

Plants outside
the quadrat
Fig. 2.4. Occurrence of plant species in a quadrat.

OBSERVATION AND RESULT

Table 2.2. Different plant species and their
population density occurring in a given area
S Plant No. of individuals
No. Total no.of Total no. of Total no. of
species per quadrat Population
individuals quadrats in quadrats
III in all the which
density
studied (B) N/B
quadrats species
studied occurred
(N) (A)
1
2
3
4
5

6
7
8.

PRECAUTIONS
1 The measurement of quadrats
should be accurate.
2 The string or cord used should not be
very thick.
3. One individual of a species
should be counted only once in the
quadrat.
CoreExperiments 27

VIVA VOCE
Q.1. Define population.
Ans. Thesum
totall of all the individuals of a species occuring in an area constitutethe population
of that species.
0.2. What ispopulation density ?
Ans It is the number of individuals per unit area of a species.
0.3. Whya quadrat of large size is generally further divided into smaller units?
Ans. In smaller units, the individuals of different species can be counted more accurately.
0.4. What isdemography ?
Ans. Statistical study of human population is called demography.
Q.5. What is natality?
Ans. Natality is the birth rate of a species or the rate by which new individuals are added to a
population by birth.
Q.6. What is mortality?
Ans. Mortality is the death rate or the rate by which individuals die from apopulation.
Q.7. What is a quadrat?
Ans. Aquadrat is asample plot ofaspecific size used for the study of population or acommunity.
 EXE IM
Study ofI Plant
3AENT
Population
INTRODUCTION
) The percentage

speies occurring in the quadrat.

() The percentage frequency is
calculated as follows:
Frequencyde termined
frequency of different plant species in a population can be
laying quadrats/segments of suitable size and recording the
number of
individuals of eacbyh
Percentage frequency
Total number of
quadrats/segments in which species occurred
Total number of x 100
quadrats/segments studied
EXPERIMENT 3.1
AIM: To study percentage
frequency of different plant species ofa given area.
REQUIREMENTS
Same in Experiment 2.1.
as
PROCEDURE
Same as in Experiment 2.1.

OBSERVATION AND RESULT

Table 3.1. Different plant
species and their percentage
S. Plant frequency occurring in a given area
No. No. of individuals
species
per quadrat Total no. of Total no. of Total no. of
II III IV
individuals
in all the quadrats in quadrats
Frequency
which percentage
quadrats studied (B) A/Bx 100
studied
species
1 (N)
Occurred
(A)
2
3.
CoreExperiments
29

5.

6
7

Calculate the percentage frequency of each species using following formula.

Total No. of quadrats in which species occurred
Percentage Frequency = x 100
Total No. of quadrats studied
PRECAUTIONS
Same as in Experiment 2.1.

VIVA VOCE
0.1. What is percent frequency of a plant species in a given area?
Ans. It is a relative term indicating, how much a particular species frequent in different quadrats/
segments, with respect of their species.
.2. What does a high frequency of a species in a particular area indicate?
Ans. It indicate the fitness of the species (i.e., reproductive fitness in
Mendelian term) in that
particular area.
0.3. At what level of ecological organisation natural selection fora
particular trait of a
species take place?
Ans. Natural selection for a particular trait occurs at a
population level.
0.4. What is the basic unit of evolution?
Ans. Population is the basic unit of evolution.
0,5. What does the population size of species in an area
indicate?
Ans. The size of population of a species tells about its
status in a particular habitat.
 EXPE
ER Study of Mitosis in
4 MEN Onion Root Tip

INTRODUCTION
1. All cells are
produced by divisions of pre-existing cells. Continuity of lifedepends on cell division
2 There are two types of cell division-mitosis and meiosis.
Somatic cells (body
mitosis. It helps in growth, by increasing the number of cells. The germ cells cells) divide .
divide by meiosis, when they form gametes (in animals) or (reproductive cella
3.
spores (in plants).
Meiosis produces four daughter cells each with half the number of
cell (2n). Therefore, it is also called chromosomes (n) of the parent
reduction division.
species to maintain its chromosome number constant,Meiosis enables a sexually reproducing
generation after generation.
4 Each cell division consists of two
events.
(a) Karyokinesis-division of the nucleus.
(b) Cytokinesis-division of the cytoplasm.
The stage of the cell prior to division is called
5 Amitotic cell
interphase.
division can be divided into following phases:
-Prophase Appearance of chromosomes.
-Metaphase Spindle formation and arrangement of
A.Karyokinesis
chromosomes at equator.
-Anaphase Movement of daughter chromosomes
towards poles.
-Telophase Formation of daughter nuclei.
B.Cytokinesis. Division of
6 cytoplasm to form two daughter cells.
The meristematic cells located in
the root tips provide the
of mitosis, while the
anthers are mnost suitable material tomost suitable material for the study
7
The chromosomes of study meiosis.
onion root tips are usedmonocotyledonous plants are large sized and better
to study mitosis and visible, therefore,
meiosis. anthers of onion or Tradescantia are used to stuay
 31
CoeExperiments

EXPERIMENT 4.1
study various
AIM: To
prepare temporary acetocarmine stained mount of onion root tip to
stagesofmitosis.

REQUIREMENTS
needles,
, flasks/glass bottles, scissors,forceps,
Onion bulbs, conical corkedvial/tube, petridishes,: microscope,
acid, acetocarmine, distilled water, spirit lamp,
methylalcohol, acetic acid, hydrochloric
etc.
sides, coverslips, blotting paper
PROCEDURE
1 Take a medium sized bulb of onion and trim
off theold roots from its base by means ofa sharp
blade.
base touching the water.
Place the onion on a conical flask/glass bottle full of water. with its
2
Keep it for a week to grow the roots.
of 1 : 3 acetic acia
Cut 5 mm off the tips of roots and put them into a vial containing a mixture
3
and methanol. Keep for one hour. This process is
called fixation. (Cutting of root tips shouta be
tO
between 9.30 a.m.
8.00 a.m. during the summer and
done in the morning between 7.00 a.m. to
11.30 a.m. during the winter).
warming to 60°C in 1 Nhydrochloric acid tor
4 Remove 2 or 3 root tips and hydrolyse them by
15minutes.

Onion bulb Onion

Roots

-Bottle
Onion root

Beaker
Water
Water

Fig. 4.1. Method of growing onion root tips.

thoroughly in water.
5. Remove the root tips and wash them
hydrolysed root tip in adrop and placea coverslip
6. Place adrop of acetocarmineon a slide. Put one
on the root.
until
coverslip with the blunt end of a pencil or needle
7. Gently squash the root by tapping the
thin layer.,
the cells separate and spread out into a very
the coverslip.
Make sure that there are noair bubbles under
seconds.
8. Gently warm the slide over a flame for a few
locate the dividing cells. Examine the
9 Observe first under the low power of the microscope to
the microscope.
different stages of mitosis under the high power of
32
Comprekenaive
Laboratory Manual in
OBSERVATIONS
Under low power of the microscope, rectangular cells with pink
high power of the microscope following stages become nucleus. are seen
Biol gy-X
distinct: (Figs. 4.2 and 4.3) scattered. Under
Maturation zone

Zone of elongation Prophase

Meristematic zone ,Metaphase

Telophase

Anaphase
Fig. 4.2. Different stages of mitosis in the Interphase
onion root tip.
1. Interphase
) It is a non-dividing phase of the cell cycle
between two successive cell divisions.
(ii) Chromatin fibres appear in the form of a
network within the nucleus.
(ii) Nuclear envelope and nucleolus are distinct.
2. Prophase
(i) Chromatin material shortens and
condenses into thread like structures called chromosomes.
(ii) Each chromosome consists of two
chromatids, jointed at a point called centromere.
(ii) Nuclear membrane and nucleolus start
disintegration and disappear at the end of prophase.
3. Metaphase
() A bipolar spindle develops in the cell. Chromosomes become thick
and two chromatids of
each chromosome become clear.
(ii) Chromosomes become arranged at the equator of the spindle.
(iii) Each chromosome get attached to the spindle fibres at its centromere.
4. Anaphase
(i) The two sister chromatids of each chromosome separate from the
centromere and move
towards the opposite poles.
(ii) The daughter chromosomes (separated chromatids) appear V, J, Land I shapes, depending
upon the position of centromere.
5. Telophase
) The spindle disappears and the daughter chromosomes uncoil to form chromatin fibres at
the two poles.
(ii) Nuclear membrane and nucleolus reappears and two daughter nuclei appear at opposite
poles.
(ii) Cytokinesis occurs by cell plate formation between the two daughter nuclei.
 CoreExperiments 33

Nuclear
membrane
Chromatin
fibres
Nucleolus
Cellmembrane

Interphase stage
Cell wall
Nuclearis yEe
Nuclear membrane
membrane
Nucleolus Disappearing
nucleolus

Chromosomestaigio Chromosomes
Cell wallilde2
Early prophase
tols Late prophase

Spindle Daughter
fibres chromosomes
Chromatids
-Spindle fibres

Early anaphase
Metaphase stage

Cellwall Daughter cells

Chromosomes Daughter nuclei
(Chromatids)
Cell plate
Nuclear
Spindle fibres
membrane
Late anaphase
Telophase stageog
Fig. 4.3. Various stages of mitosis in
onion root tip cells.

PRECAUTIONS
1. The base of the onion
bulb should be in contact of water
Z. Root tips should be fixed in the while growing the roots.
3. The slide should be
morning between 8 to 10 A.M.
warmed gently much above the flame of the spirit
lamp.
 XPER Isolationof DNA
M5EN from Plant Materials
INTRODUCTION
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the two types of nucleic acids fouma
in living systems. DNAacts as the genetic
material in most of the organisms. RNA, though, it also ac
as a genetic material in some viruses,
mostly functions as a messenger adapter, structural and in some
cases as a catalyticmolequle.
Alhuman knowledge, especially of natural
comfort and well beingof human beings Biotechnologysciences is directed to develop technologies for the
in the twentieth century. The current break has emerged as an off shoot of modern biology
throughs in the field of biotechnology are production of
genetically modified organisms (plants, animals and micro
(r DNA) technology. organisms) through recombinant DNA
Recombinant DNA technology (Genetic engineering,
to introduce foreign DNA has allowed breeders
in other organisms, including
organisms are called Genetically Modified bacteria, yeasts, animals and plants). Such
of DNA from a variety of sources and Organisms (GMOs). Thus, r DNA technology involves isolation
formation of new Combination of DNA.

EXPERIMENT 5.1
AIM: To isolate DNA from
papaya etc.
available plant material such as spinach leaves, green pea
seeds,
REQUIREMENTS
Plant material (such as spinach
beakers, test tubes, liquid detergent, leaves, green pea seeds or green
papaya), mortar and pestle,
non-iodised
papain solution/juice of papaya/pine apple sodium chloride, distilled
juice, 95% ethanol, spool etc. water, meat tenderizer or
PREPARATION OF SOLUTIONS
Detergent salt
sodium chloridesolution
is prepared by adding 10 mL
to 90 mL of liquid detergent and 10gof
Meat tenderizer solution is
distilled water. non-iodised
water (Juice of papaya/pineprepared
apple,
by adding 5 g of
tenderizer (enzyme) to 95 mL of distilled
meat tenderizer). filtered through muslin cloth can be used as substitute for
5% NaCl solution is
distilled water. prepared by dissolving 5 g of
non-iodised sodium chloride in 100 mL of
Chilling of ethanol must be done by
night. keeping 95% ethanol in plastic bottle in
the freezer over
(oe
Experiments 35

PROCEDURE

Take5gof
the plant tissue (spinach leaf/green pea seed/green papaya) arnd grind itin the
mortar by adding 10 ml detergent, salt solution and flter it through muslin cloth.
holding
Take10mL offthe fltrate, add 3-4 mL tenderizer/papaya juice andIswirl the test tube by
thetube between the two hands to mix the contents.

Pour10 ml chilled ethanol carefully downthe side of test tubeto form allayer onthe top of
the content;let it stand undisturbed for about 3 minutes.

Using the glass rod stir gently through interface of the two layers to collect the precipitate of
DNA and place it in a test tube with 5% NaCl or distilled water.
through
The quantity of DNA present in the given plant material can be estimated
spectrophotometer.

Fig. 5.1. DNAthat separates out can be removed by spooling (spool = reel for winding yarn).

OBSERVATION
The addition of ethanol to the solution causes DNA to precipitation. The DNA fibres appears as
white precipitate of very fine threads on the glass spool.

PRECAUTIONS
1. The plant material should be washed throughly with distilled water to remove any dust and
dried by blotting before weighing.
2. Allthe glasswares used must be thoroughly cleaned and dried.
3. The chemicals and enzymes used for the experiment must be of standard quality which should
be manufactured by standard pharmaceuticals.
36
Conprekensive Laboratory Manualin

Q.1. What is biotechnology ?

VIVA VOCE
Biol gy-AN
Ans. Biotechnology deals with techniques of using live
produce products and processes useful to humans. organisms or enzymes from
Q. 2. What is recombinant DNA?
Ans. Recombinant DNA is the DNA
formed by combining DNAs from two
organistns to
organisnms. diffferent
Q.3. What is geneticengineering ?
Ans. Ifrefers to the techniques to alter
Sources|
the
these into host organisms and thus chemistry of genetic material (DNA or RNA) to
change the phenotype of the host
Q.4. What are Genetically
Modified Organisms (GMOs) ?
organisms. introduce
Ans. These are the organisms whose
genes have been altered by
Q.5. Why is the enzyme
cellulase used for isolating genetic
manipulation.
Ans. The enzyme cellulase is used to
digest the cellulosic cell wall
material from plant cells 2
Q. 6. What is the role of present in plant cells.
Ans. Detergent dissolves the
detergent in isolation of DNA?
Q.7. Why salt is added to see membranes that enclose the DNA within the cell.
DNA?
Ans. Salt water allows the DNA to
Q.8. What is the role of precipitate, when alcohol is added to the solution.
meattenderizer (enzyme) in isolation of DNA?
Ans. Meat tenderizer
(enzyme) dissolves the proteins associated with the
Q.9. Why should the DNA.
minutes? mixture after adding chilled ethanol be
allowed to stand for few
Ans. It allows more DNA to
Q. 10. Why is chilled precipitate in the alcohol layer.
Ans. The chilled condition required during the experiment?
DNA.
condition protects the DNA from cellular
enzymes and also increases the yield ot