ER Study of Pollen

M1ENT Germination

INTRODUCTION
1. Pollen grain or microspore is the first cell of male gametophyte.
2. The development of male gametophyte is precocious, ie.,it begins inside the micro- sporangium
or pollen sac.
3 The pollen grain is uninucleate in the beginning but at the time o liberation it becomes
2celled-a small generative celland alarge tube or vegetative cell.
4 On the stigma, the pollen grain absorbs water and nutrients from the stigmatic secretion
through its germ pores.
5. The tube cell gives rise to a pollen tube. The generative cell also descends into the pollen tube
and divides intotwo male gametes.

EXPERIMENT 1.1

AIM: To prepare temporary mountto observe pollengermiation ona slide.

REQUIREMENTS
Fresh seasonal ffowers, slide, coverslip, microscope, sucrose, boric acid, magnesium sulphate,
potassium nitrate, beakers etc.

PROCEDURE
1. Prepare a nutrient solution by dissolving 10g sucrose, 10 gboric acid, 30 mg magnesium
sulphate and 20 mg potassium nitrate in 100 ml of water.e
2. Take a few drops of this solution on a clean slide, and dust a few pollen grains from the stamen
of a mature flower on it.
3. Observe the slide in the microscope after 5minutes and then observe it regularly for about half
an hour.

OBSERVATION
In nutrient medium, the pollen grain germinates. The tube cell enlarges and comes out of the
pollen grain through one of the germ pores to form a pollen tube. The tube nucleus descends to the tip
of the pollen tube. Thegenerative cell also passes into it. It soon divides into two male gametes. Each
male gamete is lenticular to spherical in outline.
 Comprehenslue Loaboratory.
22
Exine
Manual in B
olloS io .buie Germ
pores
Intine

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 apollen grain ?
Ans. It is commonly globular in outline, though several other
shapes are also found.
Q2. What is palynology ?
Ans. The study of pollen grains is called
palymology.
Q.3. What is the composition of wall of pollen
Ans. It is made up of two layers, outer exine and
grain ?
inner intine.
0.4. What is the chemical nature of the
two layers of the wall of pollen grain
Ans. Intine is pecto-cellulosic in nature and
exine is made of highly resistant fatty
called sporopollenin.
0 What is tectum ?
Ans. It is the discontinuous
surface layer of the exine of the pollen
characteristic sculpturing or designs over the surface of pollen grain wall, whic
06 What is the grain.
importance
Ans. It can help a taxonomist to
of tectum toa
taxonomist ?
species. identify the pollen grains and refer them to
their fami
 Study of Plant
M2ENT Population
Density
INTRODUCTION
) Population is defined as a nearly permanent aggregation of individuals of the same kind
which inhibit a particular space or geographical area at a particular time. It is subordinate
to a species as a unit of cooperative aggregation of individuals.
() The nmber 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 agiven time is called
population density.
(iv) 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.
N
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 plant species ofa given 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 feld of 1 m
x 1m by using 3 nails and tying a string with them. Now measure 10cm on one side of
the arm of Land then the other. Prepare 10 x10sq. cm area using another piece of string.
Count the number of species occurring in this area. Increase this area to 20 x20 sq. cm and
similarly record additional species occurring in this area. Repeat the same procedure till
1x1 sq. m area covered. Record the observations as follows :
 24
Comprehenalve Laboratory
1m
Manualin Biclor
90 ovbuie
80

70

60

50

40

30

20

10

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

Using the above recorded data, prepare a

size of the quadrats on Xaxis. At one point of graph. Number of species are plotted on Yaxis and
graph, curve starts flattening or shows onlya
increase. This point denotes minimum area of the quadrant
suitable for the study of an
gradual
consideration. area under
Core Experiments 25

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 ot 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 aspecies 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 theobservation table.

50
cm

50cm

Fig. 2.3. Aquadrat.

 26 Comprekenswe Laboratory Manuallin
Biology-
Nail

Species No. 1
Species No. 2
Species No. 3
< Species No. 4
50 cm
Species No. 5
String

Nail
50 cm

Plants outside
the quadrat

Fig. 2.4. Occurrence of plant species in aquadrat.

OBSERVATION AND RESULT

Table 2.2. Different plant species and their
population density occurring ina given area
Plant No. of individuals Total no. of Total no. of Total no. of
No. species per quadrat Population
individuals quadrats in quadrats density
II III in all the which 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.
Core Experiments 27

SOVD VIVA VoCE

Q.1. Define population,
Ans. The sum total of all the individuals of a species occuring in an area constitute the population
of that species.
Q.2. What is population density ?
Ans. It is the number of individuals per unit area of a species.
Q.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.
Q.4. What is demography?bes ose sldatiueonesesdo
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 ismortality ?
Ans. Mortality is the death rate or the rate by which individuals die from a population.
Q.7. What isa quadrat ?
Ans. Aquadrat is a sample plot of a specific size used for the study of population or a community.
 CORE Study of Plan
Population
3 Frequenc
INTRODUCTION
0 The percentage frequency of different plant species in a population can be determined b
laying quadrats/segments of suitable size and recording the number of individuals of
species occuring in the quadrat. each
() The percentage frequency is calculated as follows :
Percentage frequency
Total number of quadrats/segments in which species occurred
X100
Total number of quadrats/segments studied

EXPERIMENT 3.1
AIM: To stuay percentage frequency of
different plant species of a given area.
REQUIREMENTS
Same as in Experiment 2.1.
PROCEDURE
Same as in Experiment 2.1.

OBSERVATION AND RESULT

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

1.
studied Occurred
2
(N) (A)
3
 29
Core Experiments

5
6.
7.
8

Calculate the percentage frequency of each species using following formula.

Total No. of quadrats in which species occurred x 100
Percentage Frequency = Total No. of quadrats studied

PRECAUTIONS
Same as in Experiment 2.1.
VIVA VOCE
area?
Q. 1. What is percent frequency of a plant species in a given quadrats/
species frequent in different
Ans. It is a relative term indicating, how much a particular
segments, with respect of their species.
2. What does a high frequency ofa species in a particular area indicate?
Q.
indicate the fitness of the species (i.e., reproductive fitness in Mendelian term) in that
Ans. It
particular area.
level of ecological organisation natural selection for a particular trait
Q.3. At what
species take place?
occursat a population level.
Ans. Natural selection fora particular trait
evolution?
Q.4. What is the basic unit of
evolution.
Ans. Population is the basic unit of indicate?
What does the population size of species in an area
Q. 5.
population of a species tells about its status in a particular habitat.
Ans. The sizeof
CORE
Study of Mitosis in
Onion Root Tip
4
INTRODUCTION
1. Allcels are produced by divisions of pre-existing cells. Continuity of life depends on cell division
2. There are two types of cell division--mitosis and meiosis. Somatic cells (body cells) divide bu
mitosis. It helps in growth, by increasing the number of cells. The germ cells (reproductive cell
divide by meiosis, when they form gametes (in animals) or spores (in plants).
3 Meiosis produces four daughter cells each with half the number of chromosomes (n) of the
cell (2n). Therefore, it is also called reduction division. parent
species tomaintain its chromosome number constant,
Meiosis enables a sexually reproducing
4 Each cell division consists of two
generation after generation.
events.
(a) Karyokinesis-division of the
nucleus.
(b)
Cytokinesis-division
The stage of the cell prior to
of the cytoplasm.
5. Amitotic cell
division is called interphase.
division can be divided into following phases :
-Prophase Appearance of chromosomes.
A. Karyokinesis -Metaphase Spindle formation and
chromosomes at equator.arrangement
of
-Anaphase Movement of daughter chromosomes
towards poles.
B.
Cytokinesis. LTel
Division ophase
of
- Formation of daughter nuclei.
6. The
meris
of mitosis, te matic cells cytoplasm to form two
located the root tips daughter cells.
in
while the anthers are most provide the most suitable
7. The suitable material for the study
chromosomes of material to study meiosis.
onion root
meiosis.
tips are
monocotyledonous
used to plants are
study mitosis and anthers large sized and better
of onion or visible, therefore,
Tradescantia areusedto study
 31
Core Experiments

EXPERIMENT 4. 1
various
AIM: To prepare temporary acetocarmine stained mount of onion root tip to study
stages ofmitosis.

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

Onion bulb Onion

Roots

Bottle
Onion root

Water Beaker

Water

Fig. 4.1. Method of growing onion root tips.

5. Remove the root tips and wash them thoroughly in water.

6 Place a drop of acetocarmine on a slide. Put one hydrolysed root tip in a drop and place a coverslip
on the root.
7. Gently squash the root by tapping the coverslip with the blunt end of a pencil or needle until
the cells separate and spread out into a very thin layer.
Make sure that there are no air bubbles under the coverslip.
8. Gently warm the slide over a flame for a few seconds,
9 Observe first under the low power of the microscope to locate the dividing cells. Examine the
different stages of mitosis under the high power of the microscope.
32
Comprekensive Laboratory Manual in Biology-Xi
OBSERVATIONS
Under lowpower of the microscope, rectangular cells with pink nucleus are
high power of the microscope following stages become distinct : (Figs. 4.2 and 4.3)seen scattered. Undey

Maturation zone

Zone of elongation Prophase

Metaphase
Meristematic zone

Telophase

Anaphase
Interphase
Fig. 4.2. Different stages of mitosis in the onion root tip.
1. Interphase
) It is a non-dividing phase of the cell cycle
between two successive cell divisions.
ü) Chromatin fibres appear in the form of a
network within the nucleus.o
(in) Nudear envelopeand nucdeolus are distinct.
2. Prophase
(i) Chromatin material shortens and
condenses into thread like structures called chromosomes.
() Each chromosome consists of two
(ii) Nuclear membrane and nucleolus
chromatids, jointed at a point called centromere.
start disintegration and disappear at the end of
3. Metaphase prophase.
) Abipolar spindledevelops in the cell.
each chromosome become clear. Chromosomes become thick and two chromatids of
(ii) Chromosomes become
arranged at the
(ii)) Each chromosome get attached tothe equator the spindle.
of
spindle fibres at its centromere.
4. Anaphase
(i) The two sister
chromatids of each chromosome separate from the
towards the opposite poles. centromere and move
(ii) The daughter chromosomes (separated
upon the position of centromere. chromatids) appear V, J, Land Ishapes, depending
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.
(i1) Cytokinesis occurs by cell plate fornmation between the two daughter nuclei.
CoreExperiments 33

Nuclear
membrane

Chromatin
fibres

Nucleolus

Cell membrane

HOTDUGORT
Interphase stage
Cell wall| Nuclear
Nuclear membrane
membrane Disappearing
Nucleolusolos nucleolus
Chromosomes
Chromosomes
Cell wall

Early prophase obtbohot

Late prophaseoeot

Spindlewe Daughterod
chromosomes
fibres
Chromatids
-Spindle fibres

Early anaphase
Metaphase stage
Daughter cells
Cellwall
Daughter nuclei
Chromosomes
(Chromatids) Cell plate
Nuclear
Spindle fibres membrane

Late anaphase Telophase stage

Fig. 4.3. Various stages of mitosis in onion root tip cels.

PRECAUTIONS
1. The base of the onion bulb should be in contact of water while growing the roots.
2 Root tips should be fixed in the morning between 8 to 10 A.M.
3 The slide should be warmed gently much above the fame of the spirit lamp.
 eXP Isolation of DNA
M5ENT from Plant Material
INTRODUCTION
Deoxyribonucleic acid (DNA)and ribonucleic acid (RNA) are the two types of nucleic acids fo
in lhiving systems. DNA acts as the genetic material in mostof the organisms. RNA, though, it also a
as agenetic material in some viruses, mostly functions as a messenger adapter, structural and in gor
cases as a catalytic molecule,
Allhuman knowledge, especially of natural sciences is directed to develop technologies for (e
comfort and well being of human beings Biotechnology has emerged as an off shoot of modern biolo
in the twentieth century. The current break throughs in the field of biotechnology are production t
genetically modifed organisms (plants, animals and micro organisms) through recombinant DN
(r DNA) technology. Recombinant DNA technology (Genetic engineering, has allowed breeder
to introduce foreign DNA in other organisms, including bacteria, yeasts, animals and plants). Sud
organisms are called Genetically Modified Organisms (GMOs). Thus, rDNA technology involves isolatign
of DNA from a variety of sources and formation of new combination of DNA.

EXPERIMENT 5.1
AIM: To isolate DNAfrom available plant material such as spinach leaves,green pea seeds,
papaya etc.

REQUIREMENTS
Plant material (such as spinach leaves, green pea seeds or green papaya), mortar and pestle,
beakers, test tubes, liquid detergent, non-iodised sodium chloride, distilled water, meat tenderizer or
papain solution/juice of papaya/pine apple juice,95% ethanol, spool etc.
PREPARATION OF SOLUTIONS
Detergent salt solution is prepared by adding 10 mL liquid detergent and 10 gof non-iodised
sodium chloride to 90 mL of distilled water.
Meat tenderizer solution is prepared by adding 5gof tenderizer (enzyme) to 95 mL of distilled
water (Juice of papaya/pine apple, filtered through muslin cloth can be used as substitute for
meat tenderizer).
5% NaCl solution is prepared by dissolving 5 g of non-iodised sodium chloride in 100 mL of
distilled water.
Chilling of ethanol must be done by keeping 95% ethanol in plastic bottle in the freezer over
night.
CoreExperinments 35
PROCEDURE
Take 5 g of the plant tissue (spinach leaf/green pea seed/ green papaya) and grind it in the
mortar by adding 10 ml detergent, salt
solution and filter it through muslin cloth.
Take 10 mL of the fltrate, add 3-4 mL.
thetube between the two hands to tenderizer/
r/papaya juice and swirl the test tube by holding
mix the contents.
Dour 10mL chilled ethanol carefully down the side of test tube to
-he content; let it stand undisturbed for about 3 form a layer on the top or
minutes.
UÜsing the glass rod stir gently through interface of the two layers tocollect the
DNÀ and place it in a test tube with 5% NaCl or precipitate of
distilled water.
The quantity of DNA present in the given plant material can be
spectrophotometer. estimated through

Fig. 5.1. DNAthat separates out can be removed byspooling (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.
Alltheglasswares used must be thoroughly cleaned and dried.
O. The chemicals and enzymes used for the experiment must be of standard quality which should
be manufactured by standard pharmaceuticals.
 36
Comprehenslve Laboratory Manual in
VIVA VOCE Biology
Q1. at i bieechneegy?
Ans. Biotechnology deals with techniques of using
using live
live organisms or enzymes from
produce products and processes useful to humans.
Q.2. alt i ecembinant DUA 2 organism,
Ans.
Recombinant DNA is the DNA formed by
organisms. combining DNAs from two different
Q3. Whaft is geettic enginening? Source
Ans. If refers to the techniques to
alter
the
these into host organisms and thus chemistry of genetic material (DNA or RNA)to introd
Q4. hatt ane Ganially change the phenotype of the host
organisms.
Ans. These are the MotitaAOganisne (GNO)?
organisms whose genes have been altered by
Q. 5. Whgys tthe NR manipulation.
Ans. The enzyme
Q6. Whattis thR nole
lidasUlfislatiggenetic
cellulase is used to digest the cellulosic cell wall matstiall emgant al?
present in plant cells.
fdetengentin
Ans. Detergent dissolves the isalaliin ofNA?
membranes that enclose the DNA within the cell.
Ans. Salt water allows the DNA to
Q8. What is therele okmeat precipitate, when alcohol is added to the solution.
Ans. Meat tenderizer tanenitzan (enynihinnlhiom aff DNAC?
(enzyme) dissolves the proteins associated with the
DNA.
Ans. It allows more DNA to
10. Wthgyit iilkd precipitate in the alcohol layer.
Ans. The chilled canditinneinesl duiinggtheepAninent?
DNA.
condition protects the DNA from cellular
enzymes and also increases the yield o