Mscbot 510 (L)

MSCBOT-510
(L)
M. Sc. II Semester
LABORATORY PRACTICAL II
DEPARTMENT OF BOTANY
SCHOOL OF SCIENCES
UTTARAKHAND OPEN UNIVERSITY
MSCBOT-510(L)
LABORATORY PRACTICAL-II
DEPARTMENT OF BOTANY
SCHOOL OF SCIENCES
UTTARAKHAND OPEN UNIVERSITY
Phone No. 05946-261122, 261123

Toll free No. 18001804025
Fax No. 05946-264232, E. mail info@uou.ac.in
htpp://uou.ac.in
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Expert Committee
Prof. J. C. Ghildiyal Prof. G.S. Rajwar
Retired Principal Principal
Government PG College Government PG College
Karnprayag Augustmuni
Prof. Lalit Tewari Dr. Hemant Kandpal

Department of Botany School of Health Science
DSB Campus, Uttarakhand Open University
Kumaun University, Nainital Haldwani
Dr. Pooja Juyal

Department of Botany
School of Sciences
Uttarakhand Open University, Haldwani
Board of Studies
Prof. C.M. Sharma Prof. Y.S. Rawat
Department of Botany Department of Botany
HNB Garhwal (Central) University DSB Campus, Kumaun University, Nainital
Prof. R.C. Dubey Prof. P.D. Pant

Head, Deptt of Botany & Microbiology Director, School of Sciences
Gurukul Kangri University, Haridwar Uttarakhand Open University, Haldwani
Dr. Pooja Juyal

Programme Coordinator
Dr. S.N. Ojha

Assistant Professor, Department of Botany
School of Sciences, Uttarakhand Open University, Haldwani
Unit Written By: Unit No.
1. Dr. Manish Tripathi, 1, 2, 3 & 4

SSJ Campus, Kumaun University, Almora
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2. Dr. Kamal Kishor Joshi, 5, 6, 8, 10, 13, 14 & 15
LMS Government Post Graduate College, Pithoragarh
3. Dr. Manish Dev Sharma 7, 9, 11 & 12

SGRRITS University, Patel Nagar, Dehradun
Cheif Course Editor
Dr. Prabha Tewari

Associate Professor, Department of Botany
HNB Garhwal (Central) University,
Srinagar Campus, Srinagar Garhwal
Editorial Board
Dr. S.N. Ojha Dr. Pooja Juyal

Assistant Professor, Department of Botany Department of Botany
School of Sciences School of Sciences
Uttarakhand Open University, Haldwani Uttarakhand Open University, Haldwani,
Dr. Kirtika Padalia Dr. Prabha Dhondiyal

Assistant Professor (AC) Assistant Professor (AC)
Department of Botany, School of Sciences, Department of Botany, School of Sciences
Uttarakhand Open University, Haldwani Uttarakhand Open University, Haldwani
Dr. Pushpesh Joshi

Assistant Professor (AC)
Department of Botany, School of Sciences
Course Title and Code : LABORATORY PRACTICAL-II (MSCBOT-510)

ISBN No. :
Copyright : Uttarakhand Open University
Edition : 2022
Printed By : Uttrayan Publication
Published By: Uttarakhand Open University, Haldwani, Nainital-263139
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CONTENTS
BLOCK-1 CELL BIOLOGY OF PLANTS PAGE NO.
Unit-1- Identification of different stages of mitosis and study of morphology of 06-16
metaphase chromosomes using Onion root meristems
Unit-2-Identification of different stages of meiosis from suitable plant material 17-29
(Onion Buds)
Unit-3-Study of mitotic index from suitable plant material 30-36
Unit-4-Techniques of preparation of permanent and semi-permanent slides 37-62
BLOCK-2 CYTOGENETICS AND PLANT BREEDING PAGE NO.
Unit-5-Demonstration of SEM and TEM 64-87
Unit-6- Problems based on Genetics 88-116
Unit-7-Identification of Indian varieties of important crops 117-145
BLOCK-3 PLANT DEVELOPMENT PAGE NO.

Unit-8-Study of Cytohistological zones in the shoot apical meristem (SAM) in 147-163
sectioned and double stained slides
Unit-9-Examination in shoot apices in a monocot both in T.S. and L.S. to 164-180
understand the origin of leaf primordia
Unit-10-Study of alternate and distichous, alternate and superposed, opposite 181-193

and superposed opposite and decussate leaf arrangements and examination of
rosette plants and induction of bolting
Unit-11-Microscopical examination of vertical section of leaves 194-218

Unit-12-Study of epidermal peels of leaves to study the development and final 219-248
structure of stomata and preparation of stomatal index and Demonstration of the
effect of ABA on stomatal closure
BLOCK-4 PLANT REPRODUCTION PAGE NO.

Unit-13-Examination of modes of anther dehiscence and collection of pollen 250-262
grains for microscopic examination
Unit-14-Tests for pollen viability using stains and in-vitro germination, Pollen 263-278
germination using hanging drop and sitting drop cultures
Unit-15-Study of the primary and secondary abnormal growth in plants 279-297
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BLOCK-1 CELL BIOLOGY OF PLANTS
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UNIT-1 IDENTIFICATION OF DIFFERENT STAGES OF
MITOSIS AND STUDY OF MORPHOLOGY OF
METAPHASE CHROMOSOMES USING ONION ROOT
MERISTEMS
1.1 Objectives
1.2 Introduction
1.3 Identification of different stages of mitosis
1.4 Study of morphology of metaphase chromosomes using onion root meristem
1.5 Summary
1.6 Glossary
1.7 Self Assessment Questions
1.8 References
1.9 Suggested Readings
1.10 Terminal Questions
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1.1 OBJECTIVES
After reading this unit students will be able-

 To understand the process and different stages of mitosis
 To visualize different phases of mitosis
1.2 INTRODUCTION
Every cell has a tendency to pass its genetic information to the next generation to maintain their
gene pool and for that purpose each cell increases its number (Robert, 2005; Carter and
Fankhauser, 2010). The process by which a parent cell divides into two or more daughter cells is
called cell division (Lodish, 1999). We all know cell division is a small component of the cell
cycle.
Cell division is of two types, mitotic cell division and meiotic cell division. The mitotic cell
division occurs in somatic cells and the meiotic cell division is found in reproductive cells.
Mitotic cell division is the process of nuclear division in which duplicated chromosomes are
faithfully separated from one another, producing two nuclei, each with a complete copy of all the
chromosomes present in the original cell. It is a process where the numbers of chromosome are
reduced so that cells formed contain only one member of each pair of homologous
chromosomes.
Cell cycle can generally be divided into two phases: M-phase and Interphase. Interphase is the
largest part of the cell division. Interphase is the part in which the cell prepares for the M-phase
or division phase. This is the most important phase in the cell cycle which includes diverse
activities and these activities are the basic requirement for the next mitotic phase. Interphase is
primarily divided into three phases: G1 phase, S phase and G2 phase. S phase is the period during
which DNA synthesis and replication occurs. G1 is the gap period between the point where the
mitosis ends and DNA replication starts and G2 is the gap period between the point where the
DNA replication ends and the M-phase starts. These phases also have certain check points which
make sure that all the necessary requirements for the M-phase are complete and the whole cell
cycle is strictly regulated.
Each cell division whether it is mitosis or meiosis can further be divided into two types;
karyokinesis and cytokinesis. Karyokinesis is the division of the nuclear material which is
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completed during the prophase, pro-metaphase, metaphase, and anaphase stages. Cytokinesis is
the division of the cytoplasm which occurs during the last stage of cell division known as
telophase.
Fig.1.1. An overview of the eukaryotic cell cycle
Fig.1.2. The events of eukaryotic cell division as seen under a microscope
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The process of Mitosis is divided into four stages called as Prophase, Metaphase, Anaphase and
Telophase. These stages are mentioned below in brief:
Fig.1.3. The stages of mitosis in an animal cell (left drawings) and a plant
cell (right micro-photographs)
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Prophase: During this stage, the chromosomes super coil, condense and become visible for first
time during the cell cycle. The spindle fibers start forming. Its nuclear membrane starts
disintegrating.
Prometaphase: Begins abruptly with the breakdown of the nuclear envelop. Chromosomes can
now attach to spindle microtubules via their kinetochores and undergo active movement.
Metaphase: During this stage, the spindle fibers reach and attach to centromere of each sister
chromatid. The chromosomes align along the center plane of the cell. The nuclear membrane
disintegrates completely.
Anaphase: During this stage, the centromeres start splitting and the sister chromatids begin to
migrating towards the opposite poles of the cell.
Telophase: During this stage, the chromosomes are clustered on the either end of the cell. The
nuclear membrane starts reforming. The cell plate (new cell wall) starts to form between the two
daughter nuclei. This is followed by cytokinesis.
1.3 IDENTIFICATION OF DIFFERENT STAGES OF MITOSIS

a) Requirements
1. Onion plant with root
2. Feulgen stain
3. 1 N HCl
4. Scissors
5. Forceps
6. Razor blade
7. Pipette
8. 1.5 ml plastic tubes
9. Dissection probe with wooden back
10. Microscopic slides and cover slips
11. Water bath
12. Light Microscope
b) Methodology
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1. Take the onion plant with newly sprouted roots and cut two root tips using scissors and
transfer them into a plastic microfuge tube.
2. Fill 2/3 of the tube with 1N HCl using a dropper.
3. Place the tube in a 60°C water bath and incubate the tube for 12- 15 minutes.
4. Remove the tube from the water bath after the incubation.
5. Discard the HCl from the tube using a pipette to the running tap water.
6. Add some drops of distilled water into the tube and rinse the root. Then remove the water
from the plastic tube using the pipette. (Rinse the roots at least three times).
7. After the washing step add 2-3 drops of Feulgen stain into the tube with root tips and
incubate the roots for 12-15 minutes. (During the incubation, the very tip of the root will
begin to turn red as the DNA stains the numerous small actively dividing cells at the time).
8. After incubation remove the stain using a pipette.
9. Again rinse the root tips with distilled water. (Rinse the roots at least three times).
10. Transfer a root tip from the tube to the centre of the microscopic slide and add a drop of
water over it.
11. Take a razor blade and cut most of the unstained part of the root.
12. Cover the root tip with a cover slip and then carefully push down on the cover slide with the
wooden end of a dissecting probe. (Push hard, but do not twist or push the cover slide
sideways). The root tip should spread out to a diameter of about 0.5- 1cm.
13. Observe it under a compound microscope in 10× objective. Scan and narrow down to a
region containing dividing cells and switch to 40× for a better view.
c) An alternative methodology
1. Cut the tip 5 to 8 mm from the freshly sprouted root. Discard the rest of the root.
2. Place the cut tip on a clean microscope slide.
3. Add 2-3 drops of acetocarmine stain to the slide.
4. Warm the slide gently and intermittently over the alcohol lamp for about one minute. (Do
not allow the slide to get hot to the touch. Do not let the root dry out).
5. Cover the slide with a cover slip or lens paper.
6. Squash the slide with your thumb using a firm and even pressure. (Avoid squashing with
such force that the cover slip breaks or slides).
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7. Observe it under a compound microscope in 10× objective. Scan and narrow down to a
region containing dividing cells and switch to 40× for a better view.
1.4 STUDY OF MORPHOLOGY OF METAPHASE

CHROMOSOMES USING ONION ROOT MERISTEM
To study the chromosomes is difficult because the cell cycle is a very rapid process, and the
chromosomes can be visualized only for a few minutes during the cell cycle. This is the only
time period when the study of chromosome morphology must be completed (Karp, 2010;
Fankhause, 2010; Alberts et al., 2008). Maximum condensation of chromosomes is achieved
during only a short phase of this cycle, and is known as the metaphase. For the convenience of
studying the metaphase chromosomes a method is required to halt the cell division at metaphase.
There are some known drugs available in market which inhibit the formation of the spindle,
arrest cell division at metaphase as well as cause further chromosome condensation. This
inhibition results in the formation of super-condensed chromosomes scattered in the cytoplasm.
This state is often termed as an "exploded metaphase" or "colchicine metaphase" (c-mitosis).
Some drugs which are being used frequently for this purpose are colchicine, 8-hydroxyquinolin,
and paradichlorobenzene. Treatment of Allium with concentrated solution of colchicine for long
periods of time results in polyploidy in chromosomes.
Onion root tips are placed in dilute colchicine solutions (0.01%, 0.05%, 0.1%, or 1.0% solutions)
for five to ten minutes and then taken out and left for thirty minutes. Then squash the roots
immediately and prepare the slides. After thirty minutes colchicine metaphases can be observed.
To avoid polyploidy, lower concentrations of colchicine (e.g., 0.05%) should be used, and
treatment should be terminated within four hours after initiation.
Apart from colchicine, 8-hydroxyquinolin and 0.002 molar solution of oxyquinolin (for three to
six hours at 180 °C) are an effective mitotic inhibitor in preparing cells for chromosome
analysis.
The roots may then be squashed immediately, or killed and fixed for later use after the treatment.
A wide variety of stains is available to analyze a chromosome and one of the most used stain is
acetocarmine. The first advantage to select acetocarmine over any other stain is that it can be
applied directly to the living tissues without any pre-conditioning such as killing and fixing. The
second advantage is that it completes the requirements of both a stain and a fixing agent.
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Acetocarmine also has one disadvantage of staining the cytoplasm in many cells. This can be
overcome by fixation of the root tip cells with 1:3 acid alcohol for twelve hours before
attempting to stain the tissue.
The staining of the chromosomes can be enhanced by adding iron to the stain and this can be
easily achieved by teasing the stained tissue with the help of an iron dissecting needle. The iron
of the needle reacts with the acetic acid, causing the stain to darken.
1.5 SUMMARY
The process of cell division (M-phase of the cell cycle) consists of nuclear division (mitosis)
followed by cytoplasmic division (cytokinesis). The nuclear division is mediated by a
microtubule-based mitotic spindle, which separates the chromosomes, while the cytoplasmic
division is mediated by an actin-filament-based contractile ring. Mitosis is largely organized by
the microtubule asters that form around each of the two centrosomes produced when the
centrosome duplicates. Centrosome duplication begins during the S and G2 phases of the cell
cycle, and the duplicated centrosomes separate and move to opposite sides of the nucleus at the
onset of M phase to form the two poles of the mitotic spindle. Large membrane-bounded
organelles, such as the Golgi apparatus and the endoplasmic reticulum, break up into many
smaller fragments during M phase, which ensures their even distribution into daughter cells
during cytokinesis.
1.6 GLOSSARY
Acetocarmine stain: Carmine is a basic dye that is prepared from the insect Coccus cacti.
Dissolve 10 g carmine (Fisher C579-25) in 1 L of 45% glacial acetic acid, add boileezers, and
reflux for 24 h. Filter into dark bottles and store at 4°C. This solution can be stored for a long
time. Staining can be intensified by adding ferric chloride (FeCl 2·6H2O) by adding 5 mL of a 10
% ferric chloride solution per 100 mL of % acetocarmine.
Centromere: Constricted region of a mitotic chromosome that holds sister chromatids together.
Also the site on the DNA where the kinetochore forms that captures microtubules from the
mitotic spindles.
Colchicine: It is an alkaloid derived from the plant autumn crocus (Colchicum autumnale). It
inhibits microtubule polymerization and thus assembly of the mitotic spindle, demonstrates the
presence of another checkpoint in the cell cycle. When colchicine is added to cultured cells, the
cells enter mitosis and arrest with condensed chromosomes.
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Compound microscope: A compound microscope is an instrument that is used to view
magnified images of small objects on a glass slide. It can achieve higher levels of magnification
than stereo or other low power microscopes and reduce chromatic aberration.
DNA replication: Process by which a copy of a DNA molecule is made.
Feulgen stain: Feulgen stain is a staining technique discovered by Robert Feulgen and used in
histology to identify chromosomal material or DNA in cell specimens. It is darkly stained. It
depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be
avoided.
Kinetochores: Complex structure formed from proteins on a mitotic chromosome to which
microtubules attach. Plays an active part in the movement of chromosomes to the poles.
Light Microscope: A light microscope is an instrument that uses visible light and magnifying
lenses to examine small objects not visible to the naked eye, or in finer detail than the naked eye
allows.
Microtubules: Long hollow cylindrical structure composed of the protein tubulin. It is one of
the three major classes of filaments of the cytoskeleton.
Pipette: A pipette is a laboratory equipment commonly used in chemistry, biology and medicine
to transfer a measured volume of liquid, often as a media dispenser.
Polyploidy: It is the heritable condition of possessing more than two complete sets of
chromosomes.
Sister chromatids: Tightly linked pair of chromosomes that arise from chromosome duplication
during S phase. They separate during M phase and segregate into different daughter cells.
Spindle fibers: Spindle fibers form a protein structure that divides the genetic material in a cell.
The spindle is necessary to equally divide the chromosomes in a parental cell into two daughter
cells during both types of nuclear divisions: mitosis and meiosis. During mitosis, the spindle
fibers are called the mitotic spindle.
Water bath: A water bath is laboratory equipment made from a container filled with heated
water. It is used to incubate samples in water at a constant temperature over a long period of
time. All water baths have a digital or an analogue interface to allow users to set a desired
temperature.
1.7 SELF ASSESSMENT QUESTIONS

1.7.1 Multiple Choice Questions:
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1. Out of the following which is a DNA specific stain used for viewing mitosis in onion root tip
cells?
(a) Nigrosin stain (b) Phloroglucinol stain
(c) Carbol fuchsin stain (d) Feulgen stain
2. How many chromosomes are there in onion cells?
(a) Twenty (b) Six
(c) Seven (d) Eight
3. What should be the concentration of HCl used for softening of onion root tip cells before
staining?
(a) 1 N (b) 0.1 N
(c) 0.01 N (d) 0.001 N
3. Which plant’s root tip is most commonly used for viewing mitosis in labs?
(a) Onion (b) Potato
(c) Wheat (d) Rice
4. Out of the following which is the actively growing part of plant?
(a) Leaves (b) Bark
(c) Root tip (d) Flower bud
5. The term mitosis was first given by
(a) C. Nageli (b) W. Waldeyer
(c) Walther Flemming (d) Alexander Flemming
1.7.1 Answer Key: 1-(d), 2-(d), 3-(a), 4(a), 5-(c), 6-(c)
1.8 REFERENCES
 Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P (2008) Molecular Biology
of the Cell. 5th ed. Garland Science, New York.
 Brooker RJ (2005) Genetics: Analysis and Principles. McGraw-Hill Publishing, New York.
 Carter S and Fankhauser D. B (2010) Mitosis, Meiosis and Genetic. Genetics
 Fankhauser D.B (2008) Chromosomes in Root Tips. University of Cincinnati Clermont
College.
 https://vlab.amrita.edu/?sub=3&brch=188&sim=1102&cnt=3
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 Karp G. (2010) Cell and Molecular Biology: Concepts and Experiments. John Wiley and
Sons, Inc.
 Lodish H, Harvey, Berk A, Zipursky L, Matsudaira P, Baltimore D and Darnell J (1999)
Molecular Cell Biology. WH Freeman and Company, New York.
1.9 SUGGESTED READINGS

 Bruce A, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2008) Molecular Biology of the
Cell. Garland Science, Taylor and Francis, New York.
 Harvey L, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000) Molecular Cell
Biology. WH Freeman and Company, New York.
Sons, Inc.
1.10 TERMINAL QUESTIONS

1.10.1 Short answer type questions
1. What is a chromosome?
2. How to prepare acetocarmine dye?
3. What is metaphase?
4. What is metaphase chromosome?
5. What is mitosis and in what type of cells it occurs?
1.10.2 Long answer type questions
1. Explain cell cycle in detail?
2. State the difference between DNA, chromatin and chromosome?
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UNIT-2 UNIT-2-IDENTIFICATION OF DIFFERENT
STAGES OF MEIOSIS FROM SUITABLE PLANT
MATERIAL (ONION BUDS)
2.1 Objectives
2.2 Introduction
2.3 Identification of different stages of meiosis from suitable plant material
2.4 Summary
2.5 Glossary
2.7 References
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2.1 OBJECTIVES
 To identify various cells undergoing different stages of meiosis in onion flower buds.
2.2 INTRODUCTION
The process of producing an offspring through sexual reproduction comprises of the association
of two cells with haploid set of chromosomes. On the association of these two cells (or
fertilization) chromosome number doubles so this doubling of chromosome number is
compensated by an equivalent reduction in chromosome number at a stage prior to formation of
the gametes (Alberts et al., 2008; Carter and Fankhauser, 2010; Fankhauser 2008). This is done
by a type of cell division called meiosis. The term meiosis is coined in 1905 from the Greek
word meaning “reduction.” The meiosis type cell division assures the production of a haploid
phase during the whole life cycle, and fertilization makes sure a diploid phase (Brooker, 2005).
In the absence of meiosis there would be no reduction in the chromosome number only the
chromosome number would double with each generation, and in these circumstances sexual
reproduction would be impossible to achieve.
During meiosis, in contrast, the four chromatids of a pair of replicated homologous
chromosomes are distributed among four daughter nuclei (Karp 2010; Lodish et al., 1999).
Meiosis accomplishes this feat by incorporating two sequential divisions without an intervening
round of DNA replication (Table 2.1). In the first meiotic division, each chromosome (consisting
of two chromatids) is separated from its homologue. As a result, each daughter cell contains only
one member of each pair of homologous chromosomes. For this to occur, homologous
chromosomes are paired during prophase of the first meiotic division (prophase I, Table 2.1) by
an elaborate process that has no counterpart in mitosis (Snustad and Simmons, 2012). As they
are paired, homologous chromosomes engage in a process of genetic recombination that
produces chromosomes with new combinations of maternal and paternal alleles (metaphase I,
Table 2.1). In the second meiotic division, the two chromatids of each chromosome are separated
from one another (anaphase II, Table 2.1).
All the stages of meiosis are being described here separately:
Interphase
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Before undergoing in meiosis-I, each cell will remain in an interphase, during which the genetic
materials are duplicated due to active DNA replication.
Prophase-I
This stage is comparatively complex and can be characterized by the production in the
chromosome number followed by the no separation of chromatids. Prophase is the longest phase
and has 5 stages: leptotene, zygotene, pachytene, diplotene and diakinesis.
Leptotene (Leptos = slender, tene = band or thread)
The nuclear membrane and the nucleolus cannot be observed conspicuously. Chromosomes
appear as long thread like structure interwoven together and uniformly distributed.
Chromosomes display a beaded appearance and are called chromomeres. Ends of chromosomes
are drawn toward nuclear membrane near the centriole. In some plants, chromosomes may form
synthetic knots.
Zygotene (Zygon = paired)
The characteristic feature of this stage is the pairing of the homologous chromosomes with one
another, gene by gene, over the entire length of the chromosomes. The pairing of the
homologous chromosomes is called synapsis. Each pair of homologous chromosomes is known
as bivalent.
Pachytene (pachy = thick)
Each paired chromosomes get condensed and look shorter and thicker than in earlier sub-stages
and splits into 2 sister chromatids except at the region of the centromere. As a result of the
longitudinal division of each homologous chromosome into chromatids, there are 4 groups of
chromatids in the nucleus, this configuration is called tetrads. Crossing over take place in this
stage.
Diplotene (diplos = double)
In this stage a movement of homologous chromosomes can be seen except at the regions where
they are attached. The regions of attachment are called as chiasmata, they are the site of
exchange of parts between two homologous chromosomes. Chiasmata appear to move towards
the ends of the synapsed chromosomes in the process of terminalization.
Diakinesis (Dia = opposite, kinesis = movement or seperation)
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The chromosomes begin to coil, and appear shorter and thicker in comparison to earlier stages.
Terminalization is completed. The nucleolus detaches from the nucleolar organizer and
disappears completely. The nuclear membrane starts to disintegrate and spindle formation is in
progress.
Metaphase-I
The bivalents (pair of homologous chromosomes) orient themselves at random on the equatorial
plate. The centromere of each chromosome of a terminalized tetrad is directed toward the
opposite poles. The chromosomal microtubular spindle fibers remain attached with the
centromeres and homologous chromosomes ready to separate.
Anaphase-I
It is characterized by the separation of whole chromosomes of each homologous pair (tetrad), so
that each pole of the dividing cell receives either a paternal or maternal longitudinally double
chromosome of each tetrad. This ensures a change in chromosome number from diploid to
monoploid or haploid in the resultant reorganized daughter nuclei.
Telophase-I
The chromosomes may persist for a time in the condensed state, the nucleolus and nuclear
membrane may be reconstituted, and cytokinesis may also occur to produce 2 haploid cells.
Metaphase-II
Metaphase-II is of very short duration. The chromosomes rearrange in the equatorial plate. The
centromeres lie in the equator, while the arms are directed toward the poles. The centromeres
divide and separate into 2 daughter chromosomes.
Anaphase-II
Daughter chromosomes start migrating toward the opposite poles and the movement is brought
about by the action of spindle fibers.
Telophase-II
The chromosomes uncoil after reaching the opposite poles and become less distinct. The nuclear
membrane and nucleolus reappear, resulting in the formation of 4 daughter nuclei, which are
haploid.
Cytokinesis
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This separates each nucleus from the others. The cell wall is formed and 4 haploid cells are
produced.
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Table-2.1: Showing different stages of meiosis including key features, photographs and diagrams
S. No. MEIOSIS I Stages of Division Key Features Photographs Diagrams
PROPHASE I
1 LEPTOTENE Chromosomes, each consisting of two sister
chromatids, begin to condense.
2 ZYGOTENE Homologous chromosomes begin to pair.
3 PACHYTENE Homologous chromosomes are fully paired.
4 DIPLOTENE Homologous chromosomes separate, except at

chiasmata.
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5 DIAKINESIS Paired chromosomes condense further and
become attached to spindle fibers.
6 METAPHASE 1 Paired chromosomes align on the equatorial

plane in the cell.
7 ANAPHASE 1 Homologous chromosomes disjoin and move to

opposite poles of the cell.
8 TELOPHASE 1 Chromosome movement is completed and new

nuclei being to form.
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MEIOSIS II
9 PROPHASE II Chromosomes, each consisting of two sister
chromatids, condense and become attached to
spindle fibers.
10 METAPHASE II Chromosomes align on the equatorial plane in
each cell.
11 ANAPHASE II Sister chromatids disjoin and move to opposite
poles in each cell.
12 TELOPHASE II Chromosomes decondense and new nuclei
begin to form.
13 Cytokinesis The haploid daughter cells are separated by

cytoplasmic membranes.
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2.3 IDENTIFICATION OF DIFFERENT STAGES OF MEIOSIS
FROM SUITABLE PLANT MATERIAL (ONION BUDS)
Requirements:
1. Onion flower buds
2. Acetocarmine stain
3. Glass slides
4. Cover slips
5. Blotting paper
Procedure:
1. Select appropriate flower buds of different sizes from the inflorescence.
2. Fix them in Carnoy’s fluid, which is used as fixative.
3. Take a preserved flower bud and place it on a glass slide.
4. Separate the anthers and discard the other parts of the bud.
5. Put 1 or 2 drops of acetocarmine stain and squash the anthers.
6. Leave the material in the stain for 5 minutes.
7. Place a cover slip over them and tap it gently with a needle or pencil.
8. Warm it slightly over the flame of a spirit lamp.
9. Put a piece of blotting paper on the cover slip and apply uniform pressure with the thumb.
10. Observe the slide under the microscope for different meiotic stages.
2.4 SUMMARY
1. In sexually reproducing organisms gametes are produced as a result of meiosis. Therefore,
cell division in reproductive cells is called meiosis.
2. This two phase process divides the chromosome of diploid germ cell, generating four haploid
gametes. (These two phases are meiosis-I and meiosis-II).
3. During Prophase-I of meiosis-I the nuclear envelope begins to breakdown and nuclear
chromatin starts to condense into individual chromosome made up of two sister chromatids.
4. Prophase-I passes to various sub stages: Laptotene, Zygotene, Pachytene, Diplotene and
Diakinesis.
5. During Metaphase-I pairs of homologous chromosomes (called tetrads) move along their
microtubule attachments, so they are lined up along the metaphase plate.
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6. During Anaphase-I attachment between homologous chromosomes breakdown and
kinetochores pull the homologous chromosomes towards opposite poles.
7. The final stages of meiosis-I are Telophase-I and cytokinesis during which cells split apart
forming two daughter cells.
8. The first phase of Meiosis-II is prophase-II during which the nuclear envelope breaks down
and spindles reform.
9. In Metaphase-II, the chromosome aligns along the metaphase plate.
10. In Anaphase-II sister chromatids (considered individual chromosome after chromosome
separation) move towards opposite poles of meiotic spindle.
11. In the final stage of meiosis-II, chromosome reaches the poles, the spindle breaks down and
nuclear envelope reforms.
12. Cytokinesis produces four haploids (the haploid daughter cells) from the original diploid cell.
2.5 GLOSSARY
Carnoy’s fluid: Carnoy's solution is a fixative composed of 60% ethanol, 30% chloroform and
10% glacial acetic acid, 1gm of ferric chloride.
Diploid: Containing both members of each pair of homologous chromosomes, as exemplified by
most somatic cells. Diploid cells are produced from diploid parental cells during mitosis.
Fertilization: Fusion of haploid gametes, egg and sperm, to form the diploid zygote.
Generation: All of the offsprings that are at the same stage of descent from a common ancestor:
Mother and daughters represent two generations.
Genetic recombination: Reshuffling of the genes on chromosomes that occurs as a result of
breakage and reunion of segments of homologous chromosomes.
Haploid: Containing only one member of each pair of homologous chromosomes. Haploid cells
are produced during meiosis, as exemplified by sperm.
Homologous chromosomes: Paired chromosomes of diploid cells, each carrying one of the two
copies of the genetic material carried by that chromosome.
Inflorescence: An inflorescence is categorized on the basis of the arrangement of flowers on a
main axis and by the timing of its flowering (determinate and indeterminate).
Reproduction: Reproduction is the biological process by which new individual organisms
"offspring" are produced from their "parents".

26
1. In meiosis reduction division occurs during
(a) Meiosis-II (b) Meiosis-I
(c) Meiosis-I and meiosis-II both (d) None of the above
2. ‘Synapsis’ occurs during
(a) Leptotene of prophase-I (b) Metaphase-I
(c) Zygotene of prophase-I (d) Pachytene of prophase-I
3. During ‘pachytene’ which activity occurs?
(a) Alignment of homologous chromosomes (b) Chiasmata formation
(c) Crossing over between homologous chromosomes (d) Formation of synaptonemal complex
4. Which one of the following statements is incorrect?
(a) Chiasmata are the product of crossing over
(b) Chiasmata form during diplotene phase
(c) Contraction of chromosome is maximum and rapid during pachytene
(d) Chiasmata move towards the ends and terminalize during diakinesis
5. During Metaphase-I which one of the activities takes place?
(a) Chromosome separation
(b) Homologous centromere moving towards opposite poles
(c) Chromosome alignment at the equator
(d) Chiasmata disappear completely
6. A diploid cell produces _____ cells by meiotic division
(a) Four diploid daughter cells (b) Two diploid daughter cells
(c) Two haploid and two diploid daughter cells (d) Four haploid daughter cells
7. During Prophase-II spindle reformation occurs with the help of
(a) Microtubules (b) Centromere
(c) Endoplasmic reticulum (d) None of these
8. Which one of the following is longest meiotic phase?
(a) Prophase-I (b) Metaphase-I
(c) Metaphase-II (d) Prophase-II
9. Separation of sister chromatids occurs during
(a) Metaphase-II (b) Anaphase-II
27
(c) Anaphase-I (d) Metaphase-I
10. Separation of homologous chromosomes occur during
(a) Anaphase-I (b) Metaphase-II
(c) Metaphase-I (d) Prophase-II
2.6.1Answer Key: 1-(b), 2-(c), 3-(c), 4-(c), 5-(c), 6-(d), 7-(a), 8-(a), 9-(b), 10-(a)
2.7 REFERENCES
 Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P (2008) Molecular Biology of
the Cell. 5th ed. Garland Science, New York.
 Brooker RJ (2005) Genetics: Analysis and Principles. McGraw-Hill Publishing, New York.,
 Carter JLS, Fankhauser DB (2010) Mitosis, Meiosis and Genetics.
 Fankhauser D.B (2008) Chromosomes in Root Tips. University of Cincinnati Clermont
College.
Sons, Inc.
 Snustad DP, Simmons MJ (2012) Genetics. Sixth edition, John Wiley and sons Singapore.

 Alberts B, Johnson A, Lewis J, Raff M, Roberts K and Walter P (2008) Molecular Biology
of the Cell. 5th ed. Garland Science, New York.
Sons, Inc.
 Snustad DP, Simmons MJ (2012) Genetics. Sixth edition, John Wiley and sons Singapore.

2.9.1 Short answer type questions:
1. Write a brief account of meiosis-I.
2. Write a short note on synaptonemal complex.
3. What are the chiasmata? Mention their significance.
4. Write the activity of Anaphase-I.
28
5. Write the basic differences between meiosis-I and meiosis-II.
6. What is the importance of meiosis?
7. What is a bivalent?
8. What is the number of daughter cells produced at the end of meiosis?
2.9.2 Long answer type questions:
1. Summarize the events of first meiotic prophase.
2. Describe the complete process of second meiotic division.
3. Difference between meiosis and mitosis. What is the significance of meiosis?
UNIT-3 STUDY OF MITOTIC INDEX FROM SUITABLE

PLANT MATERIAL
29
3.1 Objectives
3.2 Introduction
3.3 Study of mitotic index from suitable plant material
3.4 Summary
3.5 Glossary
3.7 References
3.1 OBJECTIVES
 To calculate the mitotic index from onion root tip
30
3.2 INTRODUCTION
The growth and development of every organism depends on the capacity of its cells to divide and
multiply (Campbell, 1983). But not all cells have the same amount of capacity of division. Some
cells take part in the process of cell division very actively and on the other hand some cells lose
the capacity to divide as they mature or divide only rarely. For example, as plant roots grow,
cells near the tip of the root, in the apical meristem, divide rapidly to push the root through the
soil. The root cap detects the pull of gravity and directs the rapid growth of cells near the tip.
So, if you want to figure out that in any plant part how many cells are in active division mode
and how many have lost the capacity to divide, then you can calculate their Mitotic Index (MI)
and you will know the exact amount of dividing cell population.
Mitotic index is defined as a ratio of the total number of dividing cells (n) and the total number
of cells (N) in a particular vision field chosen randomly under the microscope. By randomly
selecting 5 to 10 such vision fields, you can estimate the Mitotic Index for any given plant
material. With the help of Mitotic Index, you can observe that how cells differ in their ability to
divide. The mitotic index provides a measure of the capacity of cells to
divide and of the rate of cell division. It is used to identify the sites
of growth within a tissue and to determine which cell types are
dividing.
3.3 STUDY OF MITOTIC INDEX FROM SUITABLE PLANT

MATERIAL
Outline of Methodology:
A. Prepare a slide of onion root tip as describe in previous chapters.
B. Observe it under the microscope.
C. Randomly select 5-10 vision fields and take their photographs with the help of a camera.
D. Count the total number of cells present in the vision field.
E. Count the number of cells that are actually in dividing state present in the vision field.
F. Put the value in the formula of mitotic index.
All these steps are being described here in detail:
A. Slide preparation of onion root tip: Refer unit 1.
B. Observation under the microscope: Place the prepared slide under the microscope for
observation. Take a thorough survey of the whole slide.
31
C. Selection of vision field and their photographs: Select some (5-10) random vision
fields in the slide and take their 5-10 high quality photographs with a digital camera.
D. Counting the total number of cells present in the vision field: Carefully observe the
photographs taken by you and try to count the total number of cells present in the
photograph of vision fields (Figure 3.1 and 3.2; Table 3.1).
Identification of different stages of mitosis:
Here we are providing some key features to identify and differentiate the cells that in which stage
of mitosis they are:
Table 3.1: identification of the different stages of mitosis division
S. Stage of Key Features Photograph
No. Division
1 Interphase One intact nuclear region
2 Prophase Condensed chromosomes inside a nuclear

membrane
3 Metaphase Condensed chromosomes present along

the equator of the cell
32
4 Anaphase Two separate clusters of chromosomes at
two different poles of the cell
5 Telophase Two nuclear regions present within a

single cell (difficult to see as cytokinesis
occurs concurrently)
A. Count the number of cells that are actually in dividing state present in the vision
field: Now count the number of dividing cells that means the cells which are under any
stage of mitosis e.g. (Prophase, Metaphase, Anaphase or Telophase).
B. Put the value in the formula of Mitotic index: The formula of Mitotic Index is:
Where, n = Number of cells undergoing mitosis

N = Total number of cells in the vision field check for visibility
Fig.3.1: A microphotograph showing various cells (with and without visible chromosomes)
under microscope
33
Fig.3.2: A microphotograph showing counts of cells under mitosis and total number of cells
under microscope
Cells in mitosis = 20
Total number of cells = 75
Mitotic index = 20/75 = 0.267
3.4 SUMMARY
1. Mitotic index is defined as the ratio between the number of cells in a population undergoing
mitosis to the total number of cells in a population.
2. The mitotic index is a measure of cellular proliferation.
3. An elevated mitotic index indicates more cells are dividing.
4. Mitotic index is an important prognostic factor predicting both overall survival and response
to chemotherapy in most type of cancers.
5. Mitotic index is used to quantify the differences in cell division when environmental
parameters are changed.
6. Mitotic activity (and mitotic index in turn) decreases with increasing distance from the zone
of meristematic cells in the root tip.
3.5 GLOSSARY
Cell division: The process by which new cells originate from other living cells.
Chromosomes: Thread like strands that are composed of the nuclear DNA of eukaryotic cells
and are the carriers of genetic information.
34
Development: The process of an individual organism growing organically; a purely biological
unfolding of events involved in an organism changing gradually from a simple to a more
complex level.
Growth: The increase in size of a cell, organ, or organism. This may occur by cell enlargement
or by cell division.
Meristem: A group of plant cells that are capable of dividing indefinitely and whose main
function is the production of new growth. They are found at the growing tip of a root or a stem
(apical meristem); in the cambium (lateral meristem) and in grasses, also within the stem and leaf
sheaths (intercalary meristem).

1. Mitotic index considers those cells which are undergoing division in
a) Prophase and metaphase only
b) Prophase, metaphase, anaphase, telophase
c) Metaphase only
d) Metaphase and Anaphase only
2. In which of these cells mitotic index value should be higher

a) Cellular repair of the site of an injury
b) In normal growth of cells
c) In cancer cells
d) In stem cells
3. Mitotic index calculation in respect of plants depict that

a) Mitotic index decreases with increasing distance from root tip
b) Gradual decrease in cell division as it moves from the zone of cell division to zone of cell
elongation
c) Both “a” and “ b” are correct
d) Mitotic index of root tip meristematic zone and shoot tip meristematic zone is equally high
4. Mitotic activity increases in plants when
35
a) Decrease in gravity occurs
b) Increase in gravity occurs
c) Gravity does not affect mitotic activity weather it increases or decreases
d) Gravity is normal
3.6.1 Answer Key: 1-(b), 2-(c), 3-(c), 4-(a)
3.7 REFERENCES
 Campbell RD (1983) Mitotic Index. In: Lenhoff HM (ed) Hydra: Research Methods.
Springer, Boston, MA
 http://www.tiem.utk.edu/~gross/bioed/webmodules/mitoticindex.htm
 https://ib.bioninja.com.au/standard-level/topic-1-cell-biology/16-cell-division/mitotic-
index.html

 Campbell RD (1983) Mitotic Index. In: Lenhoff HM (ed) Hydra: Research Methods.
Springer, Boston, MA

1. Write down the brief account on application of mitotic index?
2. Brief account on environmental aspect of mitotic index?
1. What is mitotic index? Explain in detail?
2. What is the significance of mitotic index?
36
UNIT-4 TECHNIQUES OF PREPARATION OF
PERMANENT AND SEMI-PERMANENT
SLIDES
4.1 Objectives
4.2 Introduction
4.3 Techniques of preparation of semi-permanent slides
4.4 Techniques of preparation of permanent slides
4.5 Summary
4.6 Glossary
4.8 References
37
4.1 OBJECTIVES
After reading this unit students will be able
 To prepare semi-permanent and permanent slides of different plant materials.
4.2 INTRODUCTION
One of the major difficulties encountered in the anatomical examination of monocot and dicot
plants and taxonomic study of microscopic organisms is the frequent absence of reliable type
material, particularly of long-established taxa. Most of the early species descriptions are based
on only drawings and type specimens have not survived to the present day and it is not ideal
situation to be reliable on the drawings for the morphological characters of any organism.
Collection and preservation of materials in fixative is helpful but it has its own demerits. The
material to be studied might be wrongly named or the name tag can fade away from the bottle
and there is a probable risk of drying up of the contents if left neglected.
Material stored from long time works fine for most plant species but they are not suitable for
microscopic organisms. Living type cultures are of great value but many materials do not retain
their original morphology in cultures which are therefore of no use as type material. The most
accessible and easy-to-handle type specimen of any organism is a permanent slide on which the
particular type cell or colony can be marked. The location of the slide should then be included
with the original description and drawing of the taxon. A collection of good permanent slides of
plant material is also a valuable aid to the teaching of any branch of botany. Live cultures should
be used as much as possible to study the correct features of any particular species, but there are
many growth forms and life cycle stages which cannot be induced in culture and which do not
occur naturally in sufficient quantity or at the right time of year to enable them to be
demonstrated in the living state. These can be taught in practical classes only with the help of
permanent slides. Semi-permanent preparations, such as sealed wet mounts, glycerine mounts are
easy to prepare but rarely survive without drying up for more than a few years. The ideal
permanent slide is one in which the material is mounted in a natural or artificial resin which will
set hard.
There are various methods available to make the permanent or semi-permanent slides but we
should not be confused by looking at the variety of methods, we should select a method which is
38
suitable in completing our objective perfectly. The method should be selected keeping in mind
that which plant lineage (Angiosperm, Gymnosperm, Pteridophyta, Bryophyta or Algae) or class
of microscopic organisms (Bacteria or Fungi) you are going to study. The other thing which
should be taken into consideration while selecting a method to prepare slides is that what do you
want to study in a particular material e.g. morphology, anatomy, particular organelle in a cell,
chromosomes during cell division, etc. Here are provided some general steps to prepare slides of
a material to be examined; these steps may vary depending on the material to be studied.
Broad classification of slide preparation techniques:
The main methods of placing samples onto microscope slides are dry mount, wet mount, section
cutting, squash and staining are described here in brief (Fig 4.1):
4.2 Methods
1. Dry mount: It is the most basic technique for slide preparation. In this technique, a thin
sliced section of material is mounted on the center of the slide and covered it with a cover slip.
This method is ideal for observing airborne particles such as pollens and dust as well as dead
matter such as insect and aphid legs or antennae, hair, feathers etc.
Requirements: Object (sample/material) to be observe, blades, forceps, cover slips, glass slides,
microscope etc,
Procedure: Simply position a finely cut or sliced section on the center of the slide and place a
cover slip over the sample.
Advantages: Rapid preparation of slide.
Disadvantages: Dry mount can be prepared only for those samples which do not need water to
live.
Precautions: After staining one should gently blot the slide dry.
2. Wet mount: This is made by introducing a fluid solution on a slide, suspending an object in a
solution, and after covering the specimen and the solution with a cover slide.
Requirements: A liquid (e.g. water, brine, glycerin and immersion oil), forceps, pipette, glass
slides, cover slips and paper towels etc.
Procedure:
 Place a drop of fluid in the center of the slide
 Position sample on liquid, using forceps
39
 At an angle, place one side of the cover slip against the slide making contact with outer
edge of the liquid drop
 Lower the cover slowly, avoiding air bubbles
 Remove excess water with the paper towel
Advantages:
 Specimen fixation is not necessary.
 This technique of mount is easy and quick to prepare.
Disadvantages: This method provides a transitory window as the liquid will dehydrate and
living specimens will die.
Precautions: More water must be added under the cover glass from time to time in order to
avoid dryness.
3. Section cutting: It is a step in preparing a slide of biological material by cutting thin sections
at suitable plane for microscopic examination of preserved or fresh materials.
Requirements:: Razor, blade, wax, slide, cover slide, slides etc.
Procedure:
 Wash the material with tap water to remove the surface dust particles or microorganism.
 Cut the thin sections of the material.
Advantages: Easy and rapid preparation of slide.
Disadvantages: Does not allow serial cuts, which slows down the process.
Precautions: Exposure of the blade can cause accidents.
4. Squash technique for soft materials
Requirements: Material to be examined, a liquid (e.g. water, brine, glycerin and immersion oil),
forceps, pipette, glass slides, cover slips and paper towels, microscope etc
Procedure:
 Prepare a wet mount of material
 Place paper towel over the cover slip
 Gently press down, careful not to destroy the sample or break the cover glass
 Squash the sample
 Remove excess water
Advantages: It is rapid and simple method
Disadvantages: Possibility of cell damage is always there in squash while applying pressure.
40
Precautions: Do not squash over because material could overlap
5. Staining method: Cell staining techniques and preparation depend on the type of stain and
analysis used. One or more of the following procedures may be required to prepare a sample.
5.1. Semi permanent and temporary slide: This is very quick method requiring only a few
minutes or hours to prepare a slide. However, after examination, the slide is discarded.
Requirements: Razor, blade, wax, cover slide, slides, dyes, alcohol, glycerol etc
Procedure: Following steps are following to get the semi permanent slide.
(1) Fixation: It is the necessary process to kill tissues rapidly by precipitating proteins in fresh
plant material. The chemical which used in this process is called fixative. Various fixatives are
used in laboratory requiring on the nature of the hardness of the tissues. The most common
fixatives are use 70% alcohol and formalin.
(2) Staining: After fixation, material needs to stain to emphasize the internal difference between
the components of a tissue or organ. Usually single (cotton blue, safranin, fast green) and double
staining (safranin and fast green) are use to stain the material depending upon the requirements.
(3) Mounting: Mounting media employed for temporary preparations include water and 1,2,3-
propanetriol (glycerol) 30-50% aqueous solution.
Advantages: This is rapid and simple method. These preparations may be needed for a matter of
minutes or hours only.
Disadvantages: After examination slides cannot store for the long time.
Precautions:
 Discard the incomplete and oblique sections of the material.
 Tissues should be washed well after fixation.
 If the fixation process is not done or improperly done, tissues may not stain properly and
some types of fixative may crystallize out.
 Safranin is to be used to stain only the lignified tissues, over staining can be removed by
washing in water.
 Air bubbles must be avoided in slide preparation.
5.2 Permanent slide: If the slide is to be kept for long-term reference, for days or even years, it
must be made as a permanent preparation.
41
Requirements: sample material, water, series of alcohol/formaldehyde, filter paper, slides, cover
slips, xylene, DPX or canada balsam, paper towels, microscope etc.
Procedure: Fixation and staining have already been discussed above. These two steps are the
same in both the method of slide preparation.
Dehydration: Removal of water content from the cell is called dehydration. This step should be
done gradually until all traces of water are removed from the plant cells. In this process, the
section of the plant material get dehydrates with the series of different concentration of alcohol
(ethyl alcohol) step by step (30%, 50%, 70% and 90% and end with absolute alcohol).
Clearing: Where the dehydrating agent is immiscible with the mounting medium, it is necessary
to introduce an intermediate fluid that is miscible with both. Such a fluid is known as a clearing
agent. The main purpose of clearing is to remove all traces of alcohol, thus allowing the tissues
to be infiltrated with the Canada balsam or other mountant.
The most common clearing agent use in the plant science laboratory is 1,2-dimethylbenzene
(xylene) for small soft tissues. Apart from this clove oil and cedar-wood oil were also use (for
thick tissue). Toluene is another good clearing agent but it is a bit costly.
Mounting: Permanent preparations are obtained by enclosing tissues in solid, resiniferous inedia
such as Canada balsam, DPX. After the tissues have been cleared, they are mounted in a semi-
fluid 1,2-dimethylbenzene (xylene) balsam mixture. The 1,2-dimethylbenzene (xylene)
subsequently evaporates and the balsam hardens.
Advantages:
 Best method to preserve the slide for long time.
 Easy to carry the slide from one place to another without damaging the material
 After dehydration the tissues appear opaque and bacterial decay ultimately sets in.
Disadvantage:
 Preparation of permanent slide is a long and time taking process.
42
 It is a step by step process. If any step is not done properly, then the whole process has to
be done.
Precautions:
 If dehydration carried out too rapidly, it causes distortion and shrinkage, especially of
delicate tissues, by setting up violent diffusion currents.
 Incomplete dehydration is indicated by cloudiness in the clearing agent and the slide
should be returned to absolute alcohol.
 Alcohol is a highly flammable material. Adequate ventilation and no naked flames are
essential safely precautions.
 Xylene is inflammable and toxic and the drying process is prolonged and this mountant
discolours with time.
43
44
Fig 4.1: Common techniques of preparation of slides
45
4.3 TECHNIQUES OF PREPARATION OF SEMI-PERMANENT
SLIDES
For a quick observation and daily laboratory experimentation this method is very handy. There
are various kinds of plant materials given to students in various stages of their undergraduate and
postgraduate classes, hence to study all those materials there are some techniques are being
provided here.
Materials to be examined: Dicot and monocot plants (Stem and root)
Requirements: Sharp razor, brush, dropper, needles, watch glass, microscopic slides, cover-
slips, safranin, glycerine and compound microscope.
Procedure:
Preliminary work:
 Take 2-3cm long pieces of the material.
 Hold the material between thumb and first finger of your left hand.
 Hold the razor in the right hand with edge of the blade facing you and handle at right angle.
 Dip the top of the material in water.
 Then start cutting transverse sections as fast as possible in a watch glass containing water.
 Select the thinnest section of the material with the help of a delicate brush.
 Take a clean watch glass with water, transfer thin sections of the material.
After the preliminary work done follow the given steps i.e., fixation, staining and mounting
(1) Fixation: It is the necessary process to kill tissues rapidly by precipitating proteins in fresh
plant material. The chemical which used in this process is called fixative. Various fixatives are
used in laboratory requiring on the nature of the hardness of the tissues. The most common
fixatives are use 70% alcohol and formalin.
(2) Staining: After fixation, material needs to stain to emphasize the internal difference between
the components of a tissue or organ. Usually single (cotton blue, safranin, fast green) and double
staining (safranin and fast green) are use to stain the material depending upon the requirements.
For the staining, put a few drops of stain in the watch glass with water. Leave the material dipped
in it for 3-5 minutes. Drain off the excess stain and wash with water if necessary. Put the
thinnest stained section in the centre of the slide.
46
(3) Mounting: Mounting media employed for temporary preparations include water and 1,2,3-
propanetriol (glycerol) 30-50% aqueous solution.
For the mount of section, put a drop of glycerine over the material. Cover it with a cover slip
with the help of needle at the angle of 45°. Observe the slide under a compound microscope after
staining and mounting.
Precautions:
 Safranin is to be used to stain only the lignified tissues, over staining can be removed by
washing in water.
 Air bubbles must be avoided in the sections.
 Use only brush to transfer or to handle the sections. Do not use needles for this purpose.
 Discard the incomplete and oblique sections.
After preparing the slides by following the above mentioned methodology, observe it under the
microscope. Under the microscope you will identify the material whether it is section of a
monocot or dicot plant material, whether it is a section of a stem or a root. To identify the given
material you have to find some features in the section placed under the microscope for
observation:
Dicot Stem
 Multicellular hairs present on the epidermis.
 Hypodermis collenchymatous.
 Xylem endarch (metaxylem towards periphery and protoxylem towards centre).
 Vascular bundles are arranged in a ring.
 Vascular bundles are conjoint, collateral and open i.e. cambium is present.
Monocot Stem
 Hypodermis is sclerenchymatous.
 Cortex is not differentiated into endodermis and pericycle.
 Vascular bundles are scattered in the ground tissue.
 Vascular bundles are conjoint, collateral and closed.
 Each vascular bundle is surrounded by a bundle sheath.
 Xylem is y-shaped and metaxylem lies towards periphery.
Dicot Root
 Unicellular hairs are present on the epidermis.
47
 Hypodermis is absent.
 Vascular bundles are radial. Xylem and phloem are present on separate radii.
 Xylem and phloem bundles are less than 6.
 Protoxylem lies towards periphery and metaxylem lies towards centre.
Monocot Root
 Unicellular hairs are present on the epidermis.
 Hypodermis is absent.
 Vascular bundles are radial.
 Xylem and phloem are present on separate radii.
 Xylem or phloem bundles are more than 5.
 Metaxylem lies towards centre.
4.4 TECHNIQUES OF PREPARATION OF PERMANENT SLIDES

To prepare a permanent slide we should follow the steps mentioned step below:
Preliminary work:
 Take 2-3cm long pieces of the material.
 Hold the material between thumb and first finger of your left hand.
 Hold the razor in the right hand with edge of the blade facing you and handle at right
angle.
 Dip the top of the material in water.
 Then start cutting transverse sections as fast as possible in a watch glass containing
water.
 Select the thinnest section of the material with the help of a delicate brush.
 Take a clean watch glass with water, transfer thin sections of the material.
After the preliminary work done following steps are necessary to follow. 1. Stopping metabolic
activities or fixing: It is the first and the most significant step in the preparation of permanent
slides. This step includes the instant stoppage of all the cellular metabolic activities so that no
changes can occur after the death of the cell. The stoppage of all cellular activities can be done
with the help of some reagents with very quick reactions. The best reagents for this purpose are:
Absolute alcohols, Osmic acid etc.
2. Staining: The process of coloring of various components and parts of a tissue for purpose of
clear and absolute differentiation through use of different dyes (stains) is called staining. The
48
nature of dyes may be acidic, basic or neutral. There are various kinds of dyes are in use in
different staining methods (Table 4.1 & 4.2).
Table 4.1: Some other fixing and washing agents used in various protocols
Fixing agents Washing agents
Bouin’s fluid 70% Alcohol

Mercuric chloride Iodine + 70% Alcohol
70% Alcohol -
Acetic acid 50% Alcohol
Formaline (Formaldehyde) 70% Alcohol
Potassium dichromate Water and 0.12% Chloral hydrate
Osmic acid (Osmium tetra-oxide) Water
Usually the acidic dyes stain the cytoplasmic part and basic dyes stain the nuclear part. Most
stains can be used on fixed, or non-living cells, while only some can be used on living cells;
some stains can be used on either living or non-living cells. However, with a nuclear dye
cytoplasm may also be stained. For the purpose of undergraduates usually general staining
(single staining) is used, which may stain both nucleus and cytoplasm at the same time.
But, where specific stains are used, usually they are used in combinations of two (double
staining), three (triple staining), four or even more stains.
a) Single Staining: For the purpose of single staining the dyes used are, fast green/safranin in
70% alcoholic solution or in water.
Procedure:
 Wash the material thoroughly.
 Pass the material through the gradually increasing percentage of the solvent in which
stain is made e.g. if the stain is made in alcohol then it should first be passed through
30% alcohol, 50% alcohol and 70% alcohol for at least 3 minutes in each.
 After the saturation of the material with the solvent, put it in the stain for about 3 minutes
or till the material becomes dark red (safranin) or dark green (fast green).
 Wash the material with the solvent or with the help of acid alcohol by placing the
material for 1-2 minutes in it, and examined under microscope, till desired results are
achieved.
49
Table 4.2: Some general stains used in various staining protocols
Type of stain Used for plant material/tissue/organism Stain acquired by part of sample
Safranin (single stain) Algae Stains cellular nuclei
Cotton blue + lactophenol Fungi, yeast, moulds. Cotton blue an acid dye stains the chitin present in
the cell wall of fungi.
Lactophenol as mounting fluid.
Crystal violet + Iodine Gram positive Bacteria Bacterial cell wall (thick wall of peptidoglycon).
Safranin (as counter stain) Gram negative bacteria Bacterial cell wall (PGL is thin).
Safranin + 10% glycerine Bryophytes. Epidermis, chlorenchyma stained red.
Rhizoids are also stained but little more dark.
Glycerine as mounting fluid.
Safranin+ fast green (double Pteridophytes, gymnosperms, monocot and Thick walled cells get red stain and all the thin-
staining) dicot plant samples. walled cells get the green stain.
Aceto-orcein Usually used in order to study mitosis in Biological stain for chromosome. Colour of stain
growing root tips changes from a deep red to brownish. (Over time
stain precipitates).
Bismarck Brown Used to stain live cells, cellulose and DNA. Stains acid mucin to yellow color.
Carmine Used as staining agent in histology. Stains glycogen, mucopolysaccharides and cell
nuclei red to dark pink.
Coomassie blue Used to analyze protein (in analytical Stains proteins a brilliant blue.
biochemistry).
Commonly used to stain protein in SDS-
PAGE gels.
DAPI Used to detect DNA in plant, bacteria, and A fluorescent nuclear stain that is excited by
virus particles. ultraviolet light, showing blue fluorescence when
bound to DNA.
Eosin (Counter stain) Used in techniques of histology. Tissue stained with haematoxylin and eosin shows
Counter stain to haematoxylin. cytoplasm stained pink orange and nuclei stained
darkly, either blue or purple.
Eosin Y Used for histological analysis of plants, Stains alkaline cell parts (like cytoplasm) pink.
animals.
Ethidium bromide Used in molecular biology techniques to Commonly used as florescent tag (nucleic acid stain)
50
observe DNA sample in agarose gel. On the exposure to ultraviolet light, it will perform
florescence with an orange color.
Fuchsin Basic fuchsin used to study vascular bundle Leaves red color on vascular tissue components.
of herbaceous plants and the water
conducting system of woody plants. Used to
study mitochondria as well. Also use to stain
bacterial cell.
Hoechst stains Two types of fluorescent stains, 33258 and This is a blue florescent dye, which stains DNA and
33342. the chromatin material of the cell blue.
33258 suited for subsequent determination of
mitotic index of plant cells grown as cell
suspensions in liquid medium.
Used to observe plant cell protoplasts and
plant cell nuclei.
33342 used to observe compact chromatin of
apoptotic nuclei to identify replicating cells.
Iodine Used as a starch indicator. When in solution, starch and iodine turn a dark blue
colour.
Leishman’s stain Generally used to differentiate between and Stains nucleus of WBC blue and blood cells pink.
identify WBC, malaria parasites, and
trypanosomes.
Malachite green Used to observe endospore in bacterial Stains endospore with green colour.
population.
Methylene blue Used to highlight parts of animal, bacteria, Stains acidic cell parts (like nucleus) with blue
and blood tissue specimens. colour.
Neutral/Toluylene red Used in histology. Stains lysosomes red.
Used in living cells.
51
LABORATORY PRACTICAL-I MSCBOT-510(L)
b) Double Staining: For the purpose of double staining usually Delafleld’s Haematoxylin in
distilled water and Eosin in 70% alcohol are used.
Procedure:
 Wash the material thoroughly.
 Put the material in Haematoxylin for 5 minutes or till it turns dark or blackish blue in
color.
 After the first staining of the material it is passed through the solvent in which the second
stain is prepared e.g. since Eosin is prepared in 70% alcohol the material is passed
through 30%, 50% and 70% alcohol and is kept in Eosin for one minute.
 After staining it is just washed through a dip in 90% alcohol (never use 70% alcohol).
Advantages:
 Provides contrast by means of color that reveals structural details undetected in other slide
preparation techniques.
 With the help of double staining we can differentiate between different tissue or cellular
organization.
Precautions:
 Do not leave the material over stained because it can fool the microscopic observations.
 Carefully focus on washing the material after staining.
3. Dehydration: This process removes water from the cells and replaces it with alcohol, because
the clearing agent (e.g. Xylene or Benzene) and mounting agent and its solvent (e.g. Xylene) are
soluble in alcohol not in water.
Procedure:
 Place the material firstly in 30%, then in 50%, then in 70% and then in 90% alcohol.
 Finally leave the sample in 100% or absolute alcohol for 3-5 minutes, because if we put
the material directly in absolute alcohol it will shrink because of sudden loss of water.
Advantages:
 By dehydrating the sample we can mount our preserved preparation in a precise way.
 By dehydrating a sample of permanent preparation we can keep it for observation purpose
for long.
Precautions:
UTTARAKHAND OPEN UNIVERSITY Page 52

 The material should always be over stained and then wash it until you get your desired
color.
 After staining (single and double both) the extra stain is washed off by placing the
material in acid alcohol or acid water (depending on the solvent in which the stains were
prepared) until you achieve your desired differentiation.
 Always use either staining tube or covered cavity blocks to avoid atmospheric moisture,
excessive evaporation of alcohol and reagent and to protect the material.
4. Clearing (De-alcoholization): This step includes the replacement of dehydrating agent
(alcohol) by the solvent of mounting medium. This step is called clearing because the clearing
agent gives transparency to the cells. Cedar wood oil and Clove oil are considered as best
clearing agents but the most commonly used reagent is Xylene. As xylene makes the tissue hard
and brittle and also causes its shrinkage so it may be replaced by Cedar wood oil or Clove oil.
Procedure:
 Now place the material in any clearing agent.
 If the clearing agent turns turbid or white, it means that the dehydration is not done
perfectly.
 Place the material in absolute alcohol for 5 minutes and then in clearing agent until it
becomes transparent.
 Repeat this until the clarity achieved.
Advantage:
 De-alcoholizing agent provides transparency to cell which is a very significant thing in
slide preparation.
Precaution:
 Concentration of clearing agent should be taken as per the sample type.
5. Mounting: Now transfer the material onto a drop of mounting medium which is placed in
the centre of slide and cover it with a cover slip. Always keep in mind that the mounting
medium should be of the same Refractive Index as crown glass (R.I. = 1.5). Common
mounting mediums are: Canada balsam dissolved in xylene (1.4 refractive index), DPX,
Euparol (1.4 refractive index) etc.
Procedure:
 Put a small drop of Canada balsam in center of slide with the help of glass rod.

 Now transfer the material to this drop with the help of a brush.
 Now place the cover slip on it carefully and avoid creating an air bubble.
 Clean the extra amount of Canada balsam with the help of a blotting paper.
Advantages:
 We can physically protect the material.
 Mounting medium bonds specimen, slide and cover slip together with a clear durable
film.
6. Labelling: Now, write your name on one edge of the slide and
identification of material on the other edge of the slide and put it under
microscope for examination.
4.5 SUMMARY
Material stored from long time works fine for most plant species but they are not suitable for
microscopic organisms. Living type cultures are of great value but many materials do not retain
their original morphology in cultures which are therefore of no use as type material. To rectify
these problem slide preparation of those organisms or part of the organism is a solution. A
collection of slides of plant material is also a valuable aid to the teaching of any branch of
botany.
There are various methods available to make the permanent or semi-permanent slides but we
should not be confused by looking at the variety of methods. We should select a method which is
suitable in completing our objective perfectly. The method should be selected keeping in mind
that which plant lineage (Angiosperm, Gymnosperm, Pteridophyta, Bryophyta or Algae) or class
of microscopic organisms (Bacteria or Fungi) you are going to study. The other thing which
should be taken into consideration while selecting a method to prepare slides is that what do you
want to study in a particular material e.g. morphology, anatomy, particular organelle in a cell,
chromosomes during cell division, etc. Here are provided some general methods of preparation
of slides with their advantage, disadvantage and precautions measurements.
Method 1. Dry mount method
Used for Primarily inorganic (e.g. dust particles, dead matter, hair, feathers, pollens).
Requirement Material to be observed, Blades, brush, needles, forceps, glass slides, cover
s slips.
Procedure Simply position a finely cut or sliced section or dried pollen cells on the
center of the slide and place a cover slip over the sample.

Advantages Quick preparation.

Limitations Dry mount can be prepared only for those samples which do not need water to
live.
Precautions After staining one should gently blot the slide dry.
Method 2. Wet mount method
Used for Fresh pollen, living organisms Aquatic materials,
Requirement A liquid (e.g. water, brine, glycerin and immersion oil), forceps, pipette,
s brush, needle, glass slides, cover slips and paper towels
Procedure  Place a drop of fluid in the center of the slide
 Position sample on liquid, using forceps, brush and needle.
 At an angle, place one side of the cover slip against the slide making
contact with outer edge of the liquid drop
 Lower the cover slip slowly, avoiding air bubbles
 Remove excess water with the paper towel
Advantages Specimen fixation is not necessary so these mount are quick and easy to
prepare.
Limitations This method provides a transitory window as the liquid will dehydrate and
living specimens will die.
Precautions More water must be added under the cover glass from time to time in order to
avoid dryness.
Method 3. Section cutting method
Used for The most common important and frequent method used in plant science.
Requirement Razor, blade, wax, slide, cover slide, slides etc.
s
Procedure  Wash the material with tap water to remove the surface dust particles or
microorganism.
 Cut the thin sections of the material, cover the section with cover slip.
 Examine under microscope.
Advantages Easy and rapid preparation of slide.
Limitations Does not allow serial cuts, which slows down the process.
Precautions Exposure of the blade can cause accidents.
Method 4. Squash method
Used for Soft materials
Requirement Material to be examined, a liquid (e.g. water, brine, glycerin and immersion
s oil), forceps, pipette, glass slides, cover slips and paper towels.
Procedure  Prepare a wet mount of material
 Place paper towel over the cover slip
 Gently press down, careful not to destroy the sample or break the cover

glass
 Squash the sample and remove excess water
Advantages These are rapid and simple
Limitations Possibility of cell damage is always there in squash. While applying pressure.
Precautions Do not squash over and over, material could overlap and preparation will get
affected.
Method 5. Staining method:
Cell staining techniques and preparation depend on the type of stain and
analysis used. One or more of the following procedures may be required to
prepare a sample. Two type of staining method is used for the preparation of
slides i.e., (i) Semi permanent or temporary slide and (ii) Permanent slide
(i). Semi permanent and temporary:
Used for This is very quick method requiring only a few minutes or hours to prepare a
slide. However, after examination, the slide is discarded.
Requirement Razor, blade, wax, cover slide, slides, dyes, alcohol, glycerol etc.
s
Procedure 1. Fixation: It is the necessary process to kill tissues rapidly by precipitating
proteins in fresh plant material. The most common fixatives are use 70%
alcohol and formalin.
2. Staining: Usually single (cotton blue, safranin, fast green) and double
staining (safranin and fast green) are use to stain the material depending
upon the requirements.
3. Mounting: Mounting media employed for temporary preparations include
water and 1,2,3-propanetriol (glycerol) 30-50% aqueous solution.
Advantages This is rapid and simple method. These preparations may be needed for a
matter of minutes or hours only.
Limitations After examination slides cannot store for the long time.
Precautions  Discard the incomplete and oblique sections of the material.
 Tissues should be washed well after fixation.
 If the fixation process is not done or improperly done, tissues may not
stain properly and some types of fixative may crystallize out.
 Safranin is to be used to stain only the lignified tissues, over staining can
be removed by washing in water.
 Air bubbles must be avoided in slide preparation
(ii) Permanent slide:

Used for If the slide is to be kept for long-term reference, for days or even years, it
must be made as a permanent preparation.
Requirement Sample material, water, series of alcohol/formaldehyde, filter paper, slides,
s cover slips, xylene, DPX or canada balsam, paper towels, microscope etc.
Procedure 1. Fixation and staining: have already been discussed above. These two
steps are the same in both the method of slide preparation.
2. Dehydration: Removal of water content from the cell is called
dehydration In this process, material get dehydrates with the series of
different concentration of alcohol (ethyl alcohol: 30%, 50%, 70% and 90%
and end with absolute alcohol).
3. Clearing: Clearing is necessary to remove all traces of alcohol. The most
common clearing agent use in the plant science laboratory is 1,2-
dimethylbenzene (xylene) for small soft tissues, clove oil and cedar-wood
oil were use (for thick tissue). Toluene is another good clearing agent but
it is a bit costly.
4. Mounting: Permanent preparations are obtained by enclosing tissues in
solid, resiniferous inedia such as Canada balsam, DPX.
5. Labelling: Write the name of the material on the edge of the slide.
Advantages  Best method to preserve the slide for long time.
Limitations  Preparation of permanent slide is a long and time taking process.
Precautions  If dehydration carried out too rapidly, it causes distortion and shrinkage,
especially of delicate tissues, by setting up violent diffusion currents.
 Incomplete dehydration is indicated by cloudiness in the clearing agent
and the slide should be returned to absolute alcohol.
 Alcohol is a highly flammable. Xylene is also inflammable and toxic.
4.6 GLOSSARY
De alcoholization: This step includes the replacement of dehydrating agent (alcohol) by the
solvent of mounting medium.
Dehydration: Process to remove the water from the cell.
Double staining: Stain the material with two dyes.
Fixative: It is a chemical used in the slide preparation to make the tissues optically differentiated
by changing their refractive index value.
Labelling: Notify the slide on one edge it for identification

Mounting: A chemical used to mount microscopic specimens in slide preparations. It dries

quickly and preserves stain.
Permanent slide: If the slide is to be kept for long-term reference, for days or even years.
Semi permanent or temporary slide: This is very quick method requiring only a few minutes
or hours to prepare a slide. However, after examination, the slide is discarded.
Single staining: Stain the material with a single dye.
Staining: The process of colouring of cell or tissues with the chemical dyes (stains).

1. Which type of slide is easiest to prepare?
(a) Dry mount (b) Wet mount
(c) Prepared mount (d) Section mount
2. Name the stain which is commonly used to study plant cells:

(a) Methylene blue (b) Cotton blue
(c) Safranin (d) Acetocarmine
3. Sample dehydaration usually done by:

(a) Xylene (b) Alcohol
(c) Paper towel (d) Formalin
4. Mounting in permanent slide preparation is done by:

(a) Canada balsam (b) Glycerine and xylene
(c) DPX (d) Both a and c both
5. What is the purpose of ‘fixation’?

(a) To kill the specimen only.
(b) To place the specimen on slide gently and retain immovable till observation.
(c) To ensure cellular parts survival of specimen using chemicals.
(d) To kill the specimen and to preserve all structural and cellular element near original state as
possible.

6. DPX is more significant as mountant because:

(a) It is highly viscous.
(b) It prevents the entry of air bubble, while applying cover slip.
(c) DPX mounting dries quickly and preserves stain.
(d) Both (b) and (c).
7. Cover slip is put on the mounted material on a slide very gently to:
(a) Avoid oozing of stain. (b) Avoid oozing of glycerine.
(c) Avoid entry of air bubbles. (d) Avoid the crushing of mounting material.
8. Temporary mount of a tissue is made in:

(a) Wax (b) Alcohol
(c) Xylene (d) Glycerine
9. Which of the following is one advantage of putting liquid on a specimen as used in wet
mount slides?
(a) More permanent slide fixation. (b) Magnification of the specimen.
(c) Flattens the specimen. (d) Kills the specimen.
10. What must be added to a prepared mount slide to permanently preserve and adhere
to specimen to the slide and cover slip?
(a) Stains (b) A fixative
(c) Mordant (d) Mounting medium
4.8 REFERENCES
 Brandham, PE (1970) Techniques for the rapid preparation of permanent slides of
microscopic algae. Br. phycol. J. 5(1): 47-50.

 http://www.biologydiscussion.com/zoology/practicals/preparation-of-permanent-slides-
zoology/60158
 https://www.microscopemaster.com/microscope-slides.html
 https://biology4isc.weebly.com/slide-preparation.html.

 https://www.wikihow.com/Use-a-Microscope
 https://www.wikihow.com/Prepare-Microscope-Slides
 https://www.biologydiscussion.com/botany/practicals-botany/permanent-slide-preparations-
3-methods/54089

 Brandham, PE (1970) Techniques for the rapid preparation of permanent slides of
microscopic algae. Br. phycol. J. 5(1): 47-50.

 https://www.thoughtco.com/how-to-prepare-microscope-slides-4151127
 https://www.biologydiscussion.com/botany/practicals-botany/permanent-slide-preparations-
3-methods/54089

4.10.1Short answers type questions:
1. Give a Brief account of preparing Dry mount semi permanent slide.
2. Write down the significance of dehydration done during permanent slide preparation.
3. Give a brief account of staining.
4. Why do we wash stain after a while of applying it?
5. What could be the disadvantage of air bubble, if they enter inside cover slip over the sample
to be observed?
4.10.2 Long Answer type questions:

1. Describe the stepwise procedure of preparing permanent slide.
2. Describe the procedure of preparing semi- permanent slide; also mention account on which
they differ from permanent preparations.

BLOCK-2 CYTOGENETICS AND PLANT

BREEDING

UNIT-5 DEMONSTRATION OF SEM AND TEM
5.1 Objectives
5.2 Introduction
5.3 Demonstration of TEM
5.4 Demonstration of SEM
5.5 Summary
5.6 Glossary
5.8 References

5.1 OBJECTIVES
After reading this unit students will be able to-
 Understand about microscopy.
 Understand the difference between light and electron microscope.
 Understand the functioning of light microscope.
 Understand the functioning of electron microscope.
 Understand the uses and importance of electron microscope in field of botany.
 Understand the difference between TEM and SEM.
5.2 INTRODUCTION
Microscopy is the branch of physics more appropriately optics. In real words, it is not branch but
a technical field itself which uses different types of microscopes to view micro objects and ultra
structure of samples that cannot be possible to see with the naked or unaided eye. Thus the
technical definition of microscopy is “the use of microscope” or “the examination of minute

objects by means of a microscope”. The instrument or device which is used in microscopy is

called microscope. Microscopes are devices that are designed to produce magnified images of
small objects with the help of combination of lenses. Thus, the microscope is the scientific use of
lenses in form of combinations with their focal lengths. This group of instruments includes not
only multiple-lens designs with objectives, condenser and eyepiece, but also single lens very
simple hand-held devices such as a magnifying glass.
More than seven hundred years ago, simple glass magnifiers were developed in the form of
biconvex lens when Roger Bacon (1267) described a lens for the first time. However, his
observation was not pursued immediately thereafter. These may be assumed as first microscopes
to be used in which object (or specimen) could be magnified and focused by use of the magnifier
placed between the object and the eye. These "simple microscopes" could spread the image on
the retina by magnification through increasing the visual angle on the retina. Near the end of
sixteenth century (1590) glass polishers; father and son team of Hans and Zacharias Jansen
constructed a crude type of simple microscope by placing two lenses together, which permitted
them to see minute objects. In 1609-1610, Galileo made the first simple microscope with a
focusing device and observed the water flea through his microscope. In 1617-1619 the first
double lens microscope with a single convex objective and ocular appeared. The inventor of
which was thought to be the physicist C. Drebbel. This microscope was used to study the cells,
plant and animal tissue, and also the minute living organisms. Till then, the name microscope
had not been given to this device; the name ‘microscope’ was first proposed by Faber in 1625.
The "simple microscope" reached its highest state of perfection, during sixteenth century when
of Anton von Leeuwenhoek a Dutch businessman and microbiologists used lens in systematic
way for observation of animalcules. The image produced by such a magnifier, held close to the
observer's eye, appears as if it were on the same side of the lens as the object itself. The credit of
developing a compound microscope with multiple lenses goes to Robert Hooke of England.

Fig.5.1: Microscope of Robert Hooke sometime in the 1660s
The microscope illustrated in Fig.5.1 is a simple compound microscope fabricated by British

microscopist Robert Hooke sometime in the 1660s. The microscope is illuminated by oil lamp
and light from the lamp is diffused through the water filled reservoir and then focused onto the
specimen.
The early microscopes developed during the 17th and 18th centuries suffered from chromatic
and spherical aberration. The chromatic aberrations do not degrade image quality only, but also
hamper resolution. Resolution is the ability to distinguish two closely placed objects. Thus
magnification and resolution two important concept which determines the ability of microscope.
Magnification: Magnification is not the best measure of a microscope alone. In terms of

physics magnification is defined as "a measure of the ability of a lens or other optical
instruments to magnify, expressed as the ratio of the size of the image to that of the object". This
means ratio of image size and size of objective in actual.
In general magnification image can be calculated using the formula:
The magnification of a light microscope is calculated by multiplying the magnifying powers of

the eyepiece and the objective lens. If eyepiece has a power of 10x as in laboratory microscope
and the objective lens of 100x, then the final magnification would be 1000x (10 x 100). It means
the image would appear 1000 times larger than it actually or seen by naked eye. Light

microscopes generally have three different objective lenses to allow the slide to be viewed in
three separate manners. The compound microscopes achieve two-stage magnification where the
objective magnifies image projects into the body tube of the microscope; then eyepiece further
magnifies the projected image. Thus the total magnification equals to the magnification of the
objective multiplied by the magnification of the eyepiece:
Resolution: The function of any microscope is to enhance resolution. The microscope enlarges
the image size of an object and makes possible to view by eye. Because of the enlargement,
resolution is often confused with magnification, which refers to the size of an image. In general,
the greater the magnification, the greater the resolution, but this is not always true. There are
several practical limitations of lens design which can result in increased magnification without
increased resolution.
The resolution of an optical microscope is defined as the shortest distance between two points on
a specimen that can still be distinguished as separate entities. The limit of resolution is the
closest distance between two points at which the points still can be distinguished as separate
entities.
Resolution (r) = λ/(2NA)
Resolution (r) = 0.61λ/NA
λ= Wavelength of illuminating radiation

n = Refractive index of medium (For glass lens n=1.5)
n sin α = Numerical aperture (NA)
α= Angle of aperture
For better microscope the resolution should be minimum and for minimum resolution we can
decrease the magnitude of λ by means of using low wavelength radiation.

Numerical aperture (NA): Numerical aperture is the function of light collecting ability of
microscope. The numerical aperture of a lens is dependent upon two parameters, the angle of the
incidence of light onto the lens, and the refractive index (n) of the glass of which the lens is
composed. The angle of incidence is also known as the cone angle and 1/2 of this value is
designated by the symbol α. The quantity “n sin α” is called is called numerical aperture (NA)
and sin α is the sine of semi-angle of aperture of the objective.
Fig.5.2: Cone Angle and numerical aperture (NA) Fig.5.3: Numerical aperture of a lens
According to Snell's law the numerical aperture remains the same: n sin α1 = n sin α2
Resolving Power (RP): The ability to distinguish the closely placed point objects called
resolving power. Resolving power is inversely proportional to limit of resolution. Consequently,
most microscopists today use resolution rather than limit of resolution to measure the quality of
their lenses. Resolving power can be increased by use of lower wavelength radiation.
All electron microscopes use electromagnetic and/or electrostatic lenses to control the path of
electrons. In 1931 the German engineers Ernst Ruska and Maximillion Knoll succeeded in

magnifying and formation of image by use of electrons. This was, in retrospect, the moment of
the invention of the electron microscope but the first prototype was actually built by Ruska in
1933 and was capable of resolving to 50 nm. Electron microscopy is used when greatest
resolution is required i.e. to see ultrastructure of cells. There are two kinds of electron
microscope based on the electrons used in image formation: the transmission electron
microscope (TEM) and scanning electron microscope (SEM). In this unit you will study in detail
about transmission electron microscope and scanning electron microscope.
Table.1: Difference between Light Microscope and Electron Microscope

S. No. Particular/ Features Light Microscope Electron Microscope
1. Source of Light Beam of electrons
Illumination
2. Medium Air Vacuum
3. Lenses Glass Electromagnetic
4. Magnification ≈ 500X -1500X ≈ 1,50,000 X -2,00,000 X
5. Specimen seen Live or dead. Only dead or dried specimens
are seen.
6. Specimen Usually few minutes to Takes few days.
preparation Time hours.
7. Resolution Low ≈ 0.25µm to 0.3µm. High ≈ 0.001µm (≈ 250 times
higher than light microscope.
8. Image Coloured Image is black and white
9. Radiation No risk of radiation. There is risk of radiation.
10. Stain Specimens are stained by Specimens are coated with
colored dyes. heavy metals (Pt or Au) in
order to reflect electron.
11. Image observation Directly by eyes through Image is received in
ocular lens. fluorescent screen or
photographic plate.
12. Uses For the study of detailed Used in the study of external
gross internal structure surface, ultra structure of cell
(Suitable for anatomical and very small organisms.
observation). (Suitable for cellular or
molecular observation)
13. Cost and Low price and maintenance Very expensive to purchase
Maintenance costs. and high maintenance cost.

A. Light Microscope B. Electron Microscope

Fig.5.4: Schematic presentation and comparison of light and electron microscopy
5.3 DEMONSTRATION OF TRANSMISSION ELECTRON

MICROSCOPE (TEM)
In 1931, two German electrical engineers, Max Knoll and Ernst Ruska, succeeded in using
electron beam and electromagnetic lenses in place of light and glass lenses in microscopy thus
finally creating the first transmission electron microscope (TEM) for which Ruska was awarded
the Nobel Prize for Physics in 1986.

The transmission electron microscope (TEM) operates on the same basic principles as the light
microscope but uses electron beam and electromagnetic lenses instead of light beam and glass
lenses respectively. The electron beam posseses wave nature and its wavelength is far lesser than
light; it makes possible to get a resolution a thousand times better than with a light microscope. If
specimen is illuminated with the beam of electrons the better resolution is achieved which was
requirement for improvement in the field of microscopy.
Electrons are made to pass through the specimen and the image is formed on the fluorescent
screen, either by using the transmitted beam or by using the diffracted beam. The possibility for
high magnifications better resolution the TEM proved a valuable instrument in the field of
medical, biological and material science.
Transmission electron microscope (TEM), has three principle components or systems and an
additional component the vacuum system: (1) an electron gun, (2) the image-producing system
(3) the image-recording system. An electron gun (or electron emitter) produces
the electron beam, which is utilized for image formation. Image-producing system consists of the
series of electromagnetic coils which are called which focus the electrons passing through the
specimen (Fig.5.4). Image-recording system usually consists of fluorescent screen or CCD
(Charged Coupled devices) which produces image from transmitted electrons perceptible to the
eye. In addition, a vacuum system, consisting of pumps and their associated gauges and valves,
and power supplies are required.
Image formation
The beam of electrons from the electron gun is focused into a small, thin, coherent beam by the
use of the condenser lens. This beam is restricted by the condenser aperture, which excludes high
angle electrons. The beam then strikes the specimen and parts of it are transmitted depending
upon the thickness and electron transparency of the specimen. This transmitted portion is focused
by the objective lens into an image on phosphorescent screen or charge coupled device (CCD)
camera. Optional objective apertures can be used to enhance the contrast by blocking out high-
angle diffracted electrons. The image then passed down the column through the intermediate and
projector lenses, is enlarged all the way. The image strikes the phosphorescent screen and light
is generated, allowing the user to see the image. The darker areas of the image represent those
areas of the sample that fewer electrons are transmitted through while the lighter areas of the
image represent those areas of the sample that more electrons were transmitted through.

Sample Preparation
Materials to be observed under an electron microscope require processing to produce an
observable image. TEM is a microscopic technique in which an image is formed from the
electrons transmitted through the specimen, thus specimen preparation is crucial phase of TEM.
In TEM electron beam is passed through inside of an electron microscope under high vacuum in
order to enable the electron beam to travel in straight lines. Biological materials contain ample
amount of water on evaporation it can cause problem to travel electron beam. Following are
steps of specimen preparation in electron microscopy.
Cryofixation: The first stage in specimen preparing is the fixation, one of the most important
and critical stages. Fixing specimen under ultra low temperature is known as cryo-fixation. In
cryofixation specimen freezed rapidly to liquid nitrogen temperatures or below so that the water
forms vitreous (non-crystalline) ice.
Fixation: To prevent further deterioration sample are fixed by use of fixing chemicals. In
chemical fixation for electron microscopy, glutaraldehyde (C5H8O2) and osmium tetroxide
(OsO4) is often used to crosslink protein molecules and preserve lipids respectively.
Dehydration: Dehydration is the process of removing excess water from the samples. This is
done by use of organic solvents such as ethanol or acetone. For SEM specimens it helps in total
drying of specimen and for TEM specimens’ infiltration with resin and subsequent embedding.
Embedding and Sectioning: Embedding is the process of infiltration of specimen into resin
media (such as araldite or LR white resin), which are hardened into a block for subsequent
sectioning just like with wax for light microscopy. Then the specimen sectioned into thin slices
with the help of ultra-microtome with a glass or diamond knife.
Staining: Staining is done to get contrast among different structures in image. For staining
heavy metals like lead and uranium, etc. are used to scatter electrons and to give contrast
between different structures in image. Heavy metal staining provides electron density to the
sample which results in to interactions of electrons with illuminating electron beam, which
provides contrast in the final image.
Freeze-fracture and freeze-etch: For the TEM, it can be rotary-shadowed with evaporated
platinum at low angle (typically about 6°) in a high vacuum evaporator. A second coat of carbon,
evaporated perpendicular to the average surface plane is generally performed to improve stability
of the replica coating. The specimen is returned to room temperature and pressure, and then the

extremely fragile "shadowed" metal replica of the fracture surface is released from the
underlying biological material by careful chemical digestion with acids, hypochlorite solution or
SDS detergent. The floating replica is thoroughly washed from residual chemicals, carefully
picked up on an EM grid, dried then viewed in the TEM.
The freeze fracture technique gives en face views of the internal structure and organisation of
biological membranes like integral membrane spanning and structure of proteins, lipid bilayer
and other membrane features. The sample is now ready for imaging in SEM. According to
Bullivant and Ames (1966) and Bullivant et al., (1979) freeze fracture can be undertaken using
very simple equipment like a standard vacuum coating unit or a specialized high vacuum freeze-
fracture apparatus, with a liquid nitrogen-containing holder for specimens. The cryofixed
specimen is then fractured by simply breaking with sharp knife or by using a microtome while
maintained at liquid nitrogen temperature.
The specimen may then optionally be etched after fracturing. During etching, water molecules
are allowed to sublime from the frozen surface of the fractured specimen, by increasing the
temperature to about -95°C for a few minutes. Etching lowers the specimen ice table and exposes
the true surfaces of freeze-fractured membranes, thereby revealing membrane surface features of
interest. According to Severs and Shotton (1995) the introduction of ultrarapid freezing
techniques has made it possible to freeze a wide variety of living tissue sufficiently rapidly to
achieve good cryofixation of the surface layer of cells in the absence of chemical fixation and
glycerol cryoprotection, enabling their subsequent etching.
Sputter Coating: Sample is coated by an ultra-thin film of gold, gold/palladium, platinum,
chromium, etc. under low vacuum condition. This is done to prevent charging of the specimen by
the accumulation of static electric fields of the electron irradiation which is the requirement of
imaging. It also increases the amount of secondary electrons that can be detected from the
surface of the sample in the SEM and therefore increases the signal to noise ratio.
Working
The electron beams are generated by the electron gun and facilitated to fall over the prepared
specimen with the help of magnetic condensers/lenses. The specimen is adjusted in between the
condensing lens and the objective lens on specimen grid. The electron beam that has been
partially transmitted through the very thin specimen carries information about the structure of the
specimen. The incidence beam of electrons is partially transmitted and partially diffracted by

specimen. These beams are recombined at the E-wald sphere to form combined phase contrast
image. To increase the contrast of the formed image, an amplitude contrast has to be obtained.
This can be achieved by using the transmitting only and eliminating diffracted one. The beam is
passed through the objective condenser/lens and the aperture. In order to eliminate the diffracted
beam, the aperture is adjusted in such a way that the diffracted image would eliminated. The
final image formed by transmitted beam of electrons is only which is passed through the
projector lens for further magnification. This magnified final image is visualized on fluorescent
screen or light sensitive sensor such as a CCD (charge-coupled device) camera. The image
detected by the CCD may be displayed in real time on a monitor or computer. Such produced
image is called Bright Field Image. Transmission electron microscopes produce two-
dimensional, black and white images (Fig.5.4).
5.4 DEMONSTRATION OF SCANNING ELECTRON

MICROSCOPE (SEM)
In 1940 German physicist, Manfred von Ardenne, used an another method of TEM, and called it
scanning electron microscope (SEM) which uses secondary electrons to produce images. This is
also what makes SEM images look three dimensional because rather than projecting the electron
signature onto a single flat surface, the SEM measures electrons that travel at all angles and
combines it to form an image with variable depth.
SEMs are also more amenable to researchers because the specimen preparation process is less
involved. While specimens for TEM require extremely thin slicing and treatment with stains,
SEM specimens only need a thin layer of conductive coating such as a gold film before being
placed in the vacuum container, a process not even necessary if the electron beams are strong
enough. The elimination of destructive preparation techniques has allowed researchers to image
larger objects such as insects or small mechanical parts. The images produced show such fine
details of everyday objects in new ways that they at times seem alien. The microscope is still
expensive and needs to be precisely configured for optimal results, but overall, SEMs are easier
to use compared to other forms of electron microscopy. The scanning electron
microscope (SEM) is one of the most versatile instruments available for the
examination and analysis of the microstructure morphology and chemical

composition characterizations. It is necessary to know the basic principles of

light optics in order to understand the fundamentals of electron microscopy.
Image formation in the SEM is dependent on the acquisition of signals
produced from the electron beam and specimen interactions which are
mainly two types: elastic and inelastic interactions. An elastic interaction
causes scattering/deflection of the incident electron by the collision of
specimens’ atomic nucleus or outer shell electrons. There is negligible loss of
energy in such kind of interaction during the collision and by a wide-angle
directional change of the scattered electron. Incident electrons that are
elastically scattered through an angle of more than 90° are called BSE
(backscattered electrons), and used for imaging (Fig.5.5). Detection of BSEs,
provide both compositional and topographic information in the SEM. A BSE is
defined as one which has undergone a single or multiple scattering events
and which escapes from the surface with an energy greater than 50 eV. The
elastic collision between an electron and the specimen atomic nucleus
causes the electron to bounce back with wide-angle directional change.
Roughly 10–50% of the beam electrons are backscattered toward their
source, and on an average these electrons retain 60–80% of their initial
energy. The lost energy during collision causes excitement of spinning
electrons of sample atoms orbit. As a result, the excitation of the specimens’
atoms electrons during collision leads to the generation of secondary
electrons (SE, Fig.5.5), which can be used in image formation. Secondary
electrons are used principally for topographic contrast in the SEM, i.e., for
the visualization of surface texture and roughness. The topographical image
is dependent on how many of the secondary electrons actually reach the
detector. A secondary electron signal can resolve surface structures down to
the order of 10 nm or better. Although an equivalent number of secondary
electrons might be produced as a result of the specimen primary beam
interaction, only those that can reach the detector will contribute to the
ultimate image. Secondary electrons that are prevented from reaching the
detector will generate shadows or be darker in contrast than those regions

that have an unobstructed electron path to the detector. BSEs carry

information about features that are deep beneath the surface. In examining
relatively flat samples, BSEs can be used to produce a topographical image
that differs from that produced by secondary electrons, because some BSEs
are blocked by regions of the specimen that secondary electrons might be
drawn around. The detector for BSEs differs from that used for secondary
electrons in that a biased Faraday cage is not employed to attract the
electrons. In fact the Faraday cage is often biased negatively to repel any
secondary electrons from reaching the detector.
Fig.5.5: Production of SE and BSE

Construction: The SEM instrument is made up of two components: the electronic
console and the electron column. The electronic console is controlling system that allows
instrument adjustments like current, voltage, focusing, magnification, brightness contrast, etc.
Electron Column: The electron column generates electron beam under vacuum and condensed
to a small diameter by electromagnetic deflection coils or electromagnetic lenses, and scanned
across the surface of a specimen in the specimen chamber where secondary electron detector is
placed above the specimen grid inside the specimen chamber. The components of the electron
column are:

(1) Electron gun: Located at the top of the column where free electrons are generated by
thermionic emission from a tungsten filament at high voltage. These thermionic electrons are
primarily accelerated toward an anode after where controlled by condenser coils/lenses.
(2) Condenser lenses/coils: After the beam passes the anode it is influenced by two condenser
lenses that cause the beam to converge and pass through a focal point. What occurs is that the
electron beam is essentially focused down to 1000 times its original size. In conjunction with the
selected accelerating voltage the condenser lenses are primarily responsible for determining the
intensity of the electron beam when it strikes the specimen (Fig.5.6).
(3) Apertures: apertures are used to reduce and exclude the un-necessary electrons in the lenses
or coils. The lens aperture also determines the diameter or spot size of the beam at the specimen
and the resolution and depth of field.
(4) Scanning System: Scanning system is formed by back scattered electrons and secondary
electrons. The secondary electrons from the specimen are attracted to the detector by a positive
charge.
(5) Specimen Chamber: Specimen chamber is located at the lower portion of the column where
specimen stage and controls are located.
Vacuum System
The vacuum system provides a smooth media for travel of controlled electron beam which
requires that the electronic column be under vacuum at a pressure of at least 5x10-5 Torr.

Fig.5.6: Diagram representing functioning of SEM
Fig.5.7: A SEM in Laboratory Fig.5.8: An Image formed by SEM

(Courtesy: https://ares.jsc.nasa.gov/research/laboratories/sem.html) (Courtesy: https://en.wikipedia.org/wiki/File:Misc_pollen.jpg)
5.5 SUMMARY
1. Microscopy is the branch of physics more appropriately optics.

2. The technical definition of microscopy is “the use of microscope” or “the examination of

minute objects by means of a microscope”.
3. The instrument or device which is used in microscopy is called microscope.
4. Microscopes are devices that are designed to produce magnified images of small objects with
the help of combination of lenses.
5. More than seven hundred years ago, simple glass magnifiers were developed in the form of
biconvex lens when Roger Bacon (1267), described the lens for the first time.
6. These "simple microscopes" could spread the image on the retina by magnification through
increasing the visual angle on the retina.
7. Near the end of sixteenth century (1590) glass polishers; father and son team of Hans and
Zacharias Jansen constructed a crude type of simple microscope. In 1609-1610 Galileo made
the first simple microscope with a focusing device and observed the water flea through his
microscope.
8. The name microscope had not been given to this device; the name ‘microscope’ was first
proposed by Faber in 1625.
9. The credit of developing a compound microscope with multiple lenses goes to Robert Hooke
of England.
10. The early microscopes developed during the 17th and 18th centuries suffered from chromatic
and spherical aberration.
11. Resolution is the ability to distinguish two closely placed objects.
12. Magnification and resolution are two important concepts which determine the ability of
microscope.
13. Magnification is not the best measure of a microscope alone.
14. The magnification of a light microscope is calculated by multiplying the magnifying powers
of the eyepiece and the objective lens.
15. The function of any microscope is to enhance resolution.
16. The resolution of an optical microscope is defined as the shortest distance between two
points on a specimen that can still be distinguished as separate entities.
17. The limit of resolution is the closest distance between two points at which the points still can
be distinguished as separate entities.

18. For better microscope the resolution should be minimum and for minimum resolution we can
decrease the magnitude of λ by means of using low wavelength radiation.
19. Numerical aperture is the function of light collecting ability of microscope.
20. The ability to distinguish the closely placed point objects called resolving power.
21. Resolving power is inversely proportional to limit of resolution.
22. All electron microscopes use electromagnetic and/or electrostatic lenses to control the path of
electrons.
23. In 1931 the German engineers Ernst Ruska and Maximillion Knoll succeeded in magnifying
and formation of image by use of electrons.
24. There are two kinds of electron microscopes based on the electrons used in image formation:
the transmission electron microscope (TEM) and scanning electron microscope (SEM).
25. The transmission electron microscope (TEM) operates on the same basic principles as the
light microscope but uses electron beam and electromagnetic lenses.
26. The electron beam posses wave nature and its wavelength is far lesser than light; it makes
possible to get a resolution a thousand times better than with a light microscope.
27. Electrons are made to pass through the specimen and the image is formed on the fluorescent
screen, either by using the transmitted beam or by using the diffracted beam.
28. Transmission electron microscope (TEM), has three principle components or systems and
an additional component the vacuum system: (1) an electron gun, (2) the image-producing
system (3) the image-recording system.
29. Materials to be observed under an electron microscope require processing to produce an
observable image.
30. The steps of specimen preparation in electron microscopy are cryofixation, fixation,
dehydration, embedding and sectioning, staining.
31. The freeze fracture technique gives en face views of the internal structure and organisation of
biological membranes like integral membrane spanning and structure of proteins, lipid
bilayer and other membrane features.
32. In 1940 German physicist, Manfred von Ardenne, uses an another method of TEM, and
called it Scanning Electron Microscope (SEM) which uses secondary electrons to produce
images.

33. The scanning electron microscope (SEM) is one of the most versatile instruments available
for the examination and analysis of the microstructure morphology and chemical
composition characterizations.
34. Image formation in the SEM is dependent on the acquisition of signals produced from the
electron beam and specimen interactions which are mainly two types: elastic and inelastic
interactions.
35. The SEM instrument is made up of two components: the electronic console and the electron
column.
36. The electronic console is controlling system that allows instrument adjustments like current,
voltage, focusing, magnification, brightness, contrast, etc.
37. The electron column generates electron beam under vacuum.
38. Components of the electron column are: (1) Electron gun (2) Condenser lenses/coils (3)
Apertures (4) Scanning System (5) Specimen Chamber
5.6 GLOSSARY
Biconvex: Convex (curved) on both sides.

CCD (Charged Coupled Device): Sensors/devices used in digital photography to record still
and moving images.
Contrast: An obvious difference between two or more objects or organelles in an image.
Cryofixation: Fixation of specimens by rapid freezing at very low temperature,
Dehydration: The process of removal of water from specimens.
Device: A piece of mechanical or electronic equipment used for a particular purpose.
Electron gun: A heavy metal cathode piece or device that produces a narrow stream of electrons
on heating.
Electron microscope: A microscope that uses a accelerated electron beam as a source of
illumination:
Electron: A negatively charged subatomic particle.
Embedding: The process by which the tissues or the specimens are enclosed in a mass of the
embedding medium using a mould.
Fluorescence: Emission of higher wavelength light by absorbing lower wavelength light.

Lens: a piece of glass with curved sides for concentrating or dispersing light rays.
Magnification: Magnification is the process of enlarging the size of object.
Microscopy: Field of science viewing small things.
Resolution: The ability to see two points as two separate points.
Resolving power: Ability of a lens to show two adjacent objects as discrete entities.
Retina: The retina is the nerve layer that lines the back of the eye which senses light.
Specimen: Any object used scientific study or display.
Staining: An auxiliary technique of dying specimen with suitable dye, used in microscopy to
enhance contrast in the microscopic image.
Ultramicrotome: A microtome for cutting extremely thin sections for electron microscopy.
Wavelength: the distance between successive crests or troughs of a wave.
5.7 SELF ASESSMENT QUESTIONS

1. The electron microscope deviced by:
(a) Knoll and Ruska (b) Galileo
(c) Robert Hooke (d) Zacharias Jansen
2. Lenses used in electron Microscopy:

(a) Glass (b) Magnetic
(c) Electromagnetic (d) None of above
3. The credit of developing a compound microscope with multiple lenses goes to:
(a) Robert Hooke (b) Zacharias Jansen
(c) Robert Brown (d) Galileo
4. To distinguish the closely placed point objects called:

(a) Magnification (b) Resolution
(c) Resolving power (d) None of these
5. Source of electron beam in electron microscopy is:

(a) Thermions (b) Electron gun

(c) Photo-electrons (d) All
6. Total Magnification is =Magnification of………… x Magnification of eyepiece.

(a) Eyepiece (b) Objective
(c) Projector (d) Condensor
7. Source of illumination in Electron Microscope:

(a) Light (b) X- Rays
(c) Electrons (d) All
8. Specimen type observed under microscope:

(a) Live (b) Dead
(c) Dead hydrated (d) Both live and dead
9. Stain used in electron microscopy:

(a) Heavy metal (b) Liquid nitrogen
(c) Colored dye (d) Carbon
10. Medium of electron beam travel in TEM:

(a) Air (b) Vapor
(c) Dry air (d) Vacuum
11. For better microscope the resolution should be:

(a) Minimum (b) Maximum
(c) Moderate (d) Can’t say
12. Fixing specimen under ultra low temperature is known as:

(a) Cryo-fixation (b) Ultra fixation
(c) Shadowing (d) Spur coating
13. Medium of travel of electrons in electron microscopy:

(a) Air (b) Vacuum

(c) Cedar oil (d) Wax
14. ………. specimen used in electron microscope

(a) Live (b) Dead
(c) Both (d) Grilled
15. Use of osmium tetroxide in electron microscopy is for:

(a) Shadowing (b) Staining
(c) Fixing (d) Dehydrator
1.6.2 Fill in the blanks:

(1) The term microscope was given by _______________.
(2) There are a __________types of microscopes based on illumination.
(3) Father of microscopy is called to __________.
(4) In electron microscopy _________ lenses are used.
(5) The virtual image of electron microscope formed on ________________.
(6) The first compound microscope was crafted by _______________.
(7) Back scattered electrons are used for image formation in___________.
(8) Secondary electrons are used for image formation in __________.
(9) Medium of path of travel of electron in electron microscope is_____________.
(10) Source of electron beam in electron microscopy is ____________________.
1.6.3 True or False:

(1) Live specimen can be observed by electron microscope.
(2) Colored image formed by electron microscope.
(3) Glass lenses are used in electron microscopy.
(4) The electron microscope was devised by Knoll and Ruska.
(5) In electron microscopy black and white image is formed on fluorescent screen.
(6) The image can be seen by naked eye directly in electron microscope.
(7) Heavy metal coated specimens used in electron microscopy.

(8) The name ‘microscope’ was first proposed by Faber in 1625.

(9) The SEM instrument is made up of two components, the electronic console and the electron
column.
(10) Dehydration is the process of removing excess water from the samples.
1.6.4 Very short answer questions:

(1) Define microscope.
(2) Who is called father of microscopy?
(3) Who devised electron microscope?
(4) Define electron gun.
(5) Define electromagnetic lens.
(6) Who crafted first compound microscope?
(7) What do you mean by cryofixation?
(8) Define resolving power.
(9) Define magnification.
1.6.1 Answer key: 1-(a), 2-(b), 3-(b), 4-(c), 5-(b), 6-(b), 7-(c), 8-(b), 9-(a), 10-(d), 11-(a), 12-
(a), 13-(b), 14-(b), 15-(c).
1.6.2 Answer key: 1- Faber, 2-two, 3-Leeuwenhoek, 4-electromagnetic 5- fluorescent screen,
6- Robert hook, 7- Scanning electron microscope, 8- Scanning electron microscope, 9-Vcuume,
10- electron gun.
1.6.3 Answer key: 1-False, 2-False, 3-False, 4-True, 5-True, 6-False, 7-True, 8-True, 9-True,
10-True.
5.8 REFERENCES
 Broglie, L D 1925. Recherches sur la théorie des quanta (Researches on the quantum
theory), Thesis, Paris, 1924, Ann. de Physique 10 (3): 22. (A translation by A.F. Kracklauer
2004) (http://aflb.ensmp.fr/LDB-oeuvres/De_Broglie_Kracklauer.pdf)

 Bullivant, S, and Ames, A 1966. A simple freeze-fracture replication method for electron
microscopy. Journal of Cell Biology: 29, 435-447.
 Bullivant, S, Metcalfe, P, and Warne, KP 1979. Fine structure of yeast plasma membrane
after freeze fracturing in a simple shielded device. In J.E. Rash and C.S. Hudson, eds.
"Freeze Fracture: Methods, Artifacts and Interpretations". 141-147. Raven Press, New York.
 Severs, N J, and Shotton, D M 1995. "Rapid Freezing, Freeze Fracture and Deep Etching."
Wiley-Liss, New York.

 Croft, W J 2006. Under the Microscope: A Brief History of Microscopy; Weiss, R.J., eds.;
Series in Popular Science; World Scientific Publishing Co. Singapore, SG.
 Bradbury, S et al. 2017. Electron Microscope. Britannica Academic [Online].
http://academic. eb.com
 Bradbury, S et al. 2011. Transmission Electron Microscope (TEM). Britannica Academic
[Online]. http://academic.eb.com.
 Wilson K, and Walker J 2009. Principles and Techniques of Biochemistry and Molecular
Biology. Cambridge University Press.
 Bradbury, S et al. 2016. Scanning Electron Microscope (SEM). Britannica Academic
[Online],. http:// academic.eb.com.
5.10.1 Short answer questions:

1. Write short note on magnification.
2. Describe resolution in brief.
3. What do you understand by resolving power?
4. Describe numerical aperture.
5. Differentiate between light and electron microscope.
6. Differentiate between transmission electron microscope and scanning electron microscope.
7. Write short note on secondary electrons.
8. What do you understand by Sputter Coating?

9. Write a note on numerical aperture.

10. Define freeze fracture technique.
5.10.2 Long answer questions:

1. Describe electron microscopy in detail.
2. Differentiate between TEM and SEM.
3. Write a detailed note on Transmission Electron Microscopy.
4. Write a note about history of microscopy?
5. Describe about sample preparation in electron microscopy.
UNIT-6 PROBLEMS BASED ON GENETICS
6.1 Objectives
6.2 Introduction
6.3 Problems based on genetics
6.4 Summary
6.5 Glossary
6.7 References

6.1 OBJECTIVES
After reading this unit students will be able to:

 Understand about genetic principles.
 Understand the laws of genetics.
 To know about genetic terminology.
 Understand the gene interactions.
 Understand the concept of dominance and incomplete dominance etc.
 Understand the major genetic problems.
 To short-out genetic problems.
6.2 INTRODUCTION
Genetics is the branch of science that deals with the study of heredity and variations of inherited
characteristics. The passing of traits from one generation (parents) to another generation
(offspring) is known as heredity. Heredity and variations are controlled by genes (Factors of
Mendel) Genes are located on DNA of chromosome inside the nucleus of a cell and are strung

together in such a way that the sequence carries information, which determines various features
(phenotypic traits). Thus genetics is the science which studies how traits are passed from
generation to generation. Genes are arranged in linear fashion on chromosomes and the position
occupied by gene is called locus. Each diploid (2n) organism has a pair of similar chromosomes
(one maternal and one paternal) i.e., diploid (2n) organisms generally have two copies of each
gene which governs a character.
In the 1866, an Austrian monk Gregor John Mendel postulated theory of inheritance based on
his experimental work on garden pea (Pisum sativum) and concluded that heredity is the result of
discrete units of inheritance called factors. Based on his experimental observation he postulated
following principles:
(1) Principle of paired factors
(2) Principle of dominance
(3) Principle of segregation= Principle of purity of gametes
(4) Principle of independent assortment
Genetics which follows Mendel’s laws known as Mendelian or classical genetics and which
doesnot follow Mendelian laws known as non-Mendelian genetics. In this unit you will study
problems related to Mendelian and non-Mendelian genetics.
1. Principles of Paired Factors: According to this principle genetic characters are controlled by
unit factors that exist in pairs in individual organism (diploid). The two factors lie on the two
homologous chromosomes at the same locus. They may represent the same i.e., homologous
(e.g., TT for pure tall pea plants) or alternate expression i.e., heterozygous (e.g., Tt for hybrid
tall pea plants) of the same character. Exceptions of this postulate are haploids (n), polyploids
(3n, 4n…) and hemizygous (XY) conditions thus cannot became a law.
2. Principle of dominance: If hybridization is made between contrasting traits of a character the
F1 generation shows only one trait is called dominant and another trait that cannot express it
known as recessive. The law of dominance is not universally applicable. Exceptions:
Codominance, Incomplete dominance, pseudodominance, overdominance.
(a) Phenotypic and genotypic ratio in case of dominance (Monohybrid Cross):
A Monohybrid cross is a type of genetic cross between two individuals with homozygous
genotypes of a single character or trait, often resulting in an opposite phenotype.

In a monohybrid cross; A particular character or trait is selected, and the alleles are indicated
with certain alphabet characters. The dominant alleles are indicated with upper case letters,
whereas the recessive alleles are indicated with lower case letters. The Punnet square is set up
by listing the phenotype and genotype of the parents being crossed. The probable combination
of the genotypes is written within the Punnet square. The phenotypic and genotypic ratios of
the offsprings are determined and written down. The resulting combination is called the F1
generation.
Fig.6.1: An experiment explaining principle of dominance in monohybrid cross
Test Cross Ratio

Phenotypic ratio = 3:1
Genotypic ratio = 1:2:1
(b) Phenotypic and genotypic ratio in case of dominance (Dihybrid Cross):
A dihybrid cross is crossing between two parents that differ in two traits. A hybrid organism is
one that is heterozygous, which means that it carries two different alleles at a particular genetic
position, or locus. In a dihybrid cross, the parents have different pairs of alleles for each trait
under observation. One parent possesses homozygous dominant alleles and the other possesses

homozygous recessive alleles. The offspring, or F1 generation, produced from the genetic cross
of such individuals are all heterozygous for the specific traits. This means that all of the F 1
individuals possess a hybrid genotype and express the dominant phenotypes for each trait.
Example: When a pureline round shape and yellow colored seed pea plant (RRYY) is crossed
with pureline wrinkled shape and green colored seed (rryy), the resulting offspring (F1
generation) are all heterozygous for round shaped and yellow color seeds (RrYy).
On selfing offspring (F2 generation) exhibits a 9:3:3:1 phenotypic ratio with respect to
variations of seed color and seed shape. This ratio can be predicted by using a Punnett square to
reveal the possible outcomes of a genetic cross based on probability. In the F 2 generation, out of
the plants 9 have yellow seeds with round shapes, 3 green seeds with round shape, 3 yellow seed
color with wrinkled shape and 1 green seed with wrinkled shape. The F2 progeny produces nine
different genotypes with four different phenotypes. It is the inherited genotype that determines
the phenotype of the individual. For example, plants with genotypes (RRYY, RrYY, RRYy, or
RrYy) have round shapes with yellow seeds. Plants with genotypes (rrYY or rrYy) have
wrinkled shapes and yellow seeds. Plants with genotypes (RRyy or Rryy) have round shapes
and green seeds, while plants with the genotype (rryy) have wrinkled shapes and green seeds.
Fig.6.2: Dominance in dihybrid cross

Phenotypic ratio: (3:1)2 = (3:1) x (3:1) = 9:3:3:1

Genotypic ratio: 1:2:1:2:4:2:1:2:1

The law of dominance is not universally applicable, exceptions are codominance, incomplete
dominance etc.
Codominance: Codominance is the genetic phenomenon in which both of the alleles of a gene
express themselves in heterozygous condition i.e., neither allele is dominant or recessive and
both get expressed. For example ABO blood groups in human, sickle-cell disease, and coat
colour in cattles.
Example: (1) ABO Blood group type: People with this blood type have A and B proteins at the
same time. The ABO gene determines what blood type a person has, and everyone has two
copies of this gene, one from each parent. There are several combinations of blood types that can
result, but when a person has both an A and a B allele, it will lead to blood types visible in the
blood, AB.
Parent
A B O
Alleles
AA AB AO
A
(Blood group A) (Blood group AB) (Blood group A)
AB BB BO
B
(Blood group AB) (Blood group B) (Blood group B)
AO BO OO
O
(Blood group A) (Blood group B) (Blood group O)
Fig.6.3: Co-dominance in case of human blood group

Example: (2) Roan coat colour in Cattle: Cattle which are homozygous for a black coat allele
(BB) are black and which are homozygous for a white coat allele (bb) are white, and
heterozygous (Bb) cattle appear roan (black patches with white) due to codominance of the black
and white coat color alleles (Bb).

Fig.6.4: Co-dominance in case of coat colour in Cattle

Incomplete Dominance: When the phenotype of F1 heterozygous genotype lies between the
phenotypes of parental homozygous genotypes, is called incomplete dominance or partial
dominance, or semidominance. Incomplete dominance is when a dominant allele, can not
completely mask the effects of a recessive allele, and resulting phenotype shows appearance of
blending of both alleles. The genotypic and phenotypic ratio (1:2:1) is same in this case. Eample
flower colour in Snap dragon (Antirrhinum majus) and 4 O’clock (Mirabilis jalapa) plant.
Incomplete Dominance in Snapdragons: As an example, incomplete dominance is seen in red
and white flowered snapdragon plants. When red flowered plant is crossed with white flowered
plant pink flowered plants are obtained in F1 generation. When the F1 generation consisting of all
pink plants is allowed to cross-pollinate, the resulting plants F2 generation consist of all three
phenotypes 1/4 Red (RR): 2/4 Pink (Rr): 1/4 White (rr). The phenotypic ratio is 1:2:1 which is
equal to its genotype. When F1 generation is allowed to cross-pollinate with true breeding red
plants, the resulting F2 plants consist of red and pink phenotypes 1/2 Red (RR): 1/2 Pink (Rr)
ratio. The phenotypic ratio is 1:1. When the F1 generation is allowed to cross-pollinate with true
breeding white plants, the resulting F2 plants consist of white and pink phenotypes 1/2 White
(rr): 1/2 Pink (Rr) ratio. The phenotypic ratio is 1:1.

Fig.6.5: Incomplete Dominance in Snapdragons
3. Principle of segregation (Principle of purity of gametes): The Law of Segregation states

that every individual organism contains two alleles for each trait, and that these alleles segregate
(separate) during meiosis such that each gamete contains only one of the alleles. Alleles
segregate during gamete formation, half of them carry one allele and half carry the other. An
offspring thus receives a pair of alleles for a trait by inheriting homologous chromosomes from
the parent organisms.
4. Principle of independent assortment: Mendel's law of independent assortment states that
the alleles of two (or more) different genes assort independently of one another during the course
of inheritance. In other words, the allele one gene does not influence the inheritance pattern of
the allele of another gene. This is due to Independent behavior of genes at the time of crossing
over because
1. They are free to change their position.
2. Random fusion of gametes.
3. Independent orientation of chromosomes during meiotic metaphase I.
For example: Mendelian dihybrid cross in garden Pea (Pisum sativum):

Fig.6.6: Independent assortment in dihybrid cross

Besides the Mendelian principles we should know about some genetic terminology.
Allele: An allele is an alternative form of a given gene (Fig.6.7). An allele that produces the
same phenotype whether its paired allele is identical or different called dominant allele and
represented by uppercase letter (T), an allele that produces its characteristic phenotype only
when its paired allele is identical called recessive allele and represented by lowercase letter (t).

Fig.6.7: Allele on homologous chromosome

Multiple allelism: Three or more kinds of genes which occupy the same locus are referred to as
multiple alleles and character governed by more than two alleles called multiple allelism.
Example 1: The ABO system in humans is controlled by three alleles, usually referred to as IA,
IB, and IO (I = isohaemagglutinin). IA and IB are codominant and produce type A and type B
antigens, respectively, which migrate to the surface of red blood cells, while I O is the recessive
allele and produces no antigen. So, the blood groups arising from the different possible
genotypes are as follows:
Genotype Blood Group

IA IA A
A 0
I I A
B B
I I B
B 0
I I B
A B
I I AB
I0 I0 O
Example 2: Wings of Drosophila: In Drosophila wings are normally long. Two mutations at the
same locus cause vestigial (reduced) wings and antlered (less developed) wings. Both vestigial
and antlered are alleles of the same wild gene and also of each other and are recessive to the
normal gene. If vestigial wings represented by symbol vg, antlered wings by vg a and the normal
allele by + symbol then there are chances of following three types of wing formation in
Drosophila:
(i) Long= ++ (+/+)
(ii) Vestigial= vg vg (vg/vg)
(iii) Antlered= vga vga (vga/vga)
Example 3: Coat colour in Rabbit: The coat colour in rabbits is determined by a series of
multiple alleles. The normal coat colour is brown. Besides this albino (white) and Himalayan are

the mutant races. The Himalayan race is similar to albino but has darker nose, ear, feet and tail.
The allele of albino (a) and Himalayan (a h) occupy the same locus. Both albino and Himalayan
are recessive to their normal allele (+). A cross between an albino and Himalayan produces a
Himalayan in the F1 but not intermediate as is case of other multiple alleles.
Self-Sterility in Plants: Kolreuter (1764) described self-sterility in tobacco (Nicotiana
longiflora). East and Yarnell described that self-sterility is due to series of alleles designated as
s1, s2, s3 and s4 etc. The hybrids S1/S2 or S1/S3 or S3/S4 are self-sterile because pollen grains from
these varieties did not develop, but pollens of S 1/S2 were effective and capable of fertilization
with S3/S4.
Homozygous and Heterozygous: Organisms can be homozygous or heterozygous for a gene
(Fig. 8). Homozygous means that the organism has two copies of the same allele for a gene. An
organism can be homozygous dominant, if it carries two copies of the same dominant allele
(TT), or homozygous recessive, if it carries two copies of the same recessive allele (tt).
Heterozygous means that an organism has two different alleles of a gene (Tt).
Fig.6.8: Homozygous and heterozygous conditions

Genotype and Phenotype: Genotype is genetic constitution of an organism i.e., full hereditary
information. Phenotype is an organism's actual observed properties means external appearance
i.e., morphology, development, or behavior. An organism's genotype is a major influencing
factor in the development of its phenotype (morphology), but it is not the only one. Even two
organisms with identical genotypes normally differ in their phenotypes. The phenotype is the
product of genotype and environmental influence. The concept of phenotypic plasticity defines
the degree to which an organism's phenotype is determined by its genotype. A high level of

plasticity means that environmental factors have a strong influence on the particular phenotype
development.
Back Cross, Out cross and test cross: When F1 individuals are crossed with one of the
two parents from which they have been derived, then such a cross is called back cross. Back
cross are of two types:- out cross and test cross. When Tt (F1) is crossed with
TT (Homozygous dominant parental), it is called out cross. When Tt (F 1) is crossed with tt
(Homozygous recessive parental), it is called test cross. A test cross is a way to explore the
genotpye of F1 individuals. Early use of the test cross was as an experimental mating test used to
determine what alleles are present in the genotype. Consequently, a test cross can help to
determine whether a dominant phenotype is homozygous or heterozygous for a specific allele.
Fig.6.9: Test cross outcomes
Epistasis: Epistasis (Greek=standing upon) is the masking genetic phenomenon when the
phenotypic effect of alleles at one gene masks or inhibits the expression of alleles of another
gene. The term “epistatis” was first of all used by Bateson (1909). It is the interaction between
non allelic genes in which one gene suppresses the expression of other gene. A gene is said to be
epistatic when its presence suppresses the effect of a gene at another locus. But when two
different genes which are not alleles, both affect the same character in such a way that the
expression of one masks, inhibits or suppresses the expression of the other gene, it is called
epistasis. The gene that suppresses is said to be epistatic, and the gene which remains obscure is
hypostatic.
Monohybrid and dihybrid cross: A monohybrid cross is a breeding experiment conducted
between parents, which differ in one specific trait only. Or in other words, the parents are
heterozygous (having dissimilar alleles) at only one locus. Over here, one parent has a dominant
gene for a specific phenotypic character (e.g. tall trait), while the other has recessive gene for the
particular phenotype (e.g. dwarf trait).

Contrary to monohybrid cross, parents that differ in two traits breed in a dihybrid cross.
To be more precise, the parental organisms are heterozygous for two different traits. In this case,
one parent has dominant genes for two characters (e.g. tall plant and red flowers) and the other
parent has recessive genes for the same two characters (dwarf plant and white flowers) in the
chromosome.
6.3 PROBLEMS BASED ON GENETICS
In this section you will study genetic problem in the form of gene interaction as non-Mendelian
genetics. When expression of one gene depends on the presence or absence of another gene in an
individual, it is known as gene interaction. The interaction of genes at different loci that affect
the same character is called epistasis. The term epistasis was first used by Bateson in 1909 to
describe two different genes which affect the same character, one of which masks the expression
of other gene. The gene that masks another gene is called epistatic gene, and the gene whose
expression is masked is termed as hypostatic gene. Epistasis is also referred to as inter-genic or
inter-allelic gene interaction.
The gene interactions have several characteristics as follows:
i. This is an essential feature of gene interaction which always involves two or more genes.
ii. The epistatic genes always affect the expression of one and the same character of an
individual.
iii. The phenotype of a gene usually depends upon the presence or absence of epistatic gene. The
gene which has masking effect is called epistatic gene and the gene whose effect is masked is
known as hypostatic gene.
iv. Epistasis leads to the modification of Mendelian di-hybrid (9:3:3:1) or tri-hybrid ratio
(27:9:9:9:3:3:3:1) in F2 generation.
v. Epistasis is generally governed by dominant genes, but cases of recessive epistasis are also
shown.
Mendelian genetics does not explain all kinds of inheritance for which the phenotypic
ratios in some cases are different from Mendelian ratios (3:1 for monohybrid, 9:3:3:1 for di-
hybrid, 27:9:9:9:3:3:3:1for tri-hybrid in F2). This is because sometimes a particular allele may be
partially or equally dominant to the other or due to existence of more than two alleles or due to

lethal alleles. These kinds of genetic interactions between the alleles of a single gene are referred
to as allelic or intra- allelic interactions.
Non-allelic or inter-allelic interactions also occur where the development of single
character is due to two or more genes affecting the expression of each other in various ways.
Thus, the expression of gene is not independent of each other and dependent on the presence or
absence of other gene or genes; These kinds of deviations from Mendelian one gene-one trait
concept is known as factor hypothesis or gene-interaction. Now epistasis term is used
synonymously with almost any type of gene interaction that involves the masking of one gene by
another gene. When epistasis is operative (gene interacts) the phenotypic ratio deviates from
Mendelian ratio (3:1 for monohybrid, 9:3:3:1 for di-hybrid and 27:9:9:9:3:3:3:1for tri-hybrid) in
F2 generation. The phenomenon of two or more gene affecting expression of each other in
various ways in the development of single character of an organism known as gene
interaction/epistasis.
Types of gene interactions/epistasis: The two or more genes interact in several manners
some common interactions are as follows:
(1) Dominant epistasis (12:3:1)
(2) Recessive epistasis or supplementary gene interaction (9:3:4)
(3) Double recessive/complementary gene interaction (9:7)
(4) Inhibitory gene interaction/dominant recessive epistasis (13:3)
(5) Polymorphic gene interaction (9:6:1)
(6) Duplicate gene interaction/double dominant epistasis (15:1)
(7) Collaborative supplementary/modified gene interaction (9:3:3:1)
(8) Polymeric gene interaction (9:6:1)
1. Dominant epistasis (12:3:1): A genetic phenomenon of non allelic gene interaction in which
a dominant gene or a dominant gene pair inhibits or masks the expression of another dominant
gene or gene pair.
For example: fruit colour in summer squash
Three types of fruit colours are present in summer squash (Cucurbita pepo), viz., white,
and yellow and green. White colour is controlled by dominant gene W and yellow colour by
dominant gene Y. This white colour gene is dominant over both yellow and green.

The green fruits are produced in recessive condition (wwyy). A cross between plants
having white and yellow fruits produced F1 with white fruits. Inter-mating of F 1 plants produces
plants with white, yellow and green coloured fruits in F 2 in 12 : 3 : 1 ratio (Fig. 10) instead of
typical Mendelian dihybrid ratio (9:3:3:1).
W = white non allelic epistatic dominant gene.
Y = yellow hypostatic dominant gene.
y = green recessive gene.
Fig.6.10: Dominant epistasis in summer squash (Cucurbita pepo)

2. Recessive epistasis or supplementary gene interaction (9:3:4): A genetic phenomenon of
non allelic gene interaction in which a gene in its homozygous recessive state masks the
expression of non allelic gene or gene pair.
In supplementary gene action, the dominant allele of one gene is essential for the
development of the concerned phenotype, while the other gene modifies the expression
of the first gene. For example, the development of grain colour in maize is governed by 2
dominant genes R and P. The dominant allele R is essential for red colour production;
homozygous state of the recessive allele (rr) checks the production of red colour. The gene P is

unable to produce any colour on its own but it modifies the colour produced by the gene R from
red to purple. The recessive allele p has no effect on grain colour.
Fig.6.11: Recessive Epistasis or supplementary gene interaction in Maize Grain
3. Double recessive/complementary gene interaction (9:7): A genetic phenomenon of non

allelic gene interaction in which homozygous recessive gene inhibits the expression of other
gene and vice versa.
Or
A genetic phenomenon of non allelic gene interaction in which a gene or gene pair requires the
help of dominant non allelic gene interaction. Complete dominance at both gene pairs, but either
recessive homozygote is epistatic to the effect of the other gene.
For example: Flower colour in sweet pea (Lathyrus odoratus):
For the production of the purple flower colour both dominant C and P (complementary)
genes are necessary. Otherwise in the absence of either dominant genes (C or P) the flower colour
become white. Thus, C and P genes interact and both are essential for the purple colour expression

of flower together. Complementation between two non-allelic genes (C and P) are essential for
production of a particular or special phenotype i.e., complementary factor.
P= Purple flower colour
C= Colourless
PPCC= Purple Colour flower
PPcc= Colourless
ppCC= Colourless
Fig.6.12: Double recessive/Complementary gene interaction in Sweat Pea (Lathyrus odoratus)
4. Inhibitory gene interaction (13:3): In Inhibitory gene interaction the non-allelic

dominant gene inhibits the expression of the other non-allelic dominant gene. A genetical
phenomenon in which a gene is recessive for its own but dominant with regard to epistasis. The
gene which inhibits the expression of an allele situated at different locus is called as inhibitory
gene.
For example: Pigmentation in paddy leaves.
In paddy plants P gene is responsible for deep purple colour. But if I gene is present along with P
then expression of purple colour is inhibited and becomes green. Thus in a cross, between green
(IIpp) and purple (iiPP), gives all F 1 offspring green but in F 2 progeny, green and purple are
obtained in ratio of 13:3 instead of typical 9:3:3:1 Mendelian F2 ratio.

PPii= Purple
PPII= Green
P= purple but when interacts with I gene than inhibited by it and becomes green.
I= Green but when interacts with P gene than inhibits P gene Make it green.
Fig.6.13: Inhibitory gene interaction paddy leaves pigmentation

6. Polymorphic gene interaction (9:6:1): When two or more genes (Allelic and non-
allelic) govern any character separately, their effect is equal but when both or all genes are
present together, there phenotypic effect is increased or raised as if the effects of the two or more
genes were additive or cumulative. In this case both or all genes shows complete dominance.
“Additive or cummulative effect of genes present at different loci is called polymerism.” Thus,
polymeric gene interaction modifies the typical 9:3:3:1 Mendelian ratio in to 9:6:1 ratio.
For example: pericarp colour in Wheat.
C1C1C2C2 = Deep Red
C1C1c2C2 = Light Red
c1c1c2c2 = Brown Colour

Fig.6.14: Polymorphic Gene interaction in wheat pericarp
7. Collaborative supplementary/modified gene interaction (9:3:3:1): A genetic

phenomenon in which two non allelic dominant gene produces the different and independent
effect but when they comes together they produces different/new effect.
They are two nonallelic genes which not only are able to produce their own effects
independently when present in the dominant state but can also interact to form a new trait.
For example: Comb types in poultry are an example of collaborative supplementary
genes, P and R. When homozygous pea combed and homozygous rose combed birds are crossed,
all the offspring of F1 generation have walnut comb. On selfing the walnut combed, F 2 generation
comes to have all the four types of combs in the ratio of 9 (walnut): 3 (pea): 3 (rose): 1 (single)
(Fig.6.15). This type of gene interaction produces the typical di-hybrid ratio of 9:3:3:1 in F 2 for a
single character. Evidently the concerned character is governed by two genes showing complete
dominance.
As worked out by Bateson and Punnett (1908), when both dominant alleles are present
‘walnut’ phenotype appears and when both recessive alleles are present ‘single’ comb appears.
‘Rose’ and ‘Pea’ phenotypes appear due to the presence of different single dominant alleles. If

pea (rrPP) and rose (RRpp) are crossed, F1 birds showed ‘walnut’ comb as it has the dominant
alleles of both the genes P and R.
Fig.6.15: Collaborative supplementary/Modified gene interaction in comb types in poultry
6.4 SUMMARY
1. Genetics is the branch of science that deals with the study of heredity and variations of
inherited characteristics.
2. Heredity and variations are controlled by genes.
3. Each diploid (2n) organism has a pair of similar chromosomes (one maternal and one
paternal) i.e., diploid (2n) organism generally have two copies of each gene which govern a
character.
4. In the 1866, an Austrian monk Gregor John Mendel postulated theory of inheritance based on
his experimental work on garden pea (Pisum sativum) and concluded that heredity is the
result of discrete units of inheritance called factors.

5. Genetics which follows Mendels laws is known as Mendelian or classical genetics and which
doesnot follow Mendelian laws is known as non-Mendalian genetics.
6. According to Mendelian principle genetic characters are controlled by unit factors that exist
in pairs in individual organism (diploid).
7. Mendel postulated four principles: 1. Principle of paired factors 2.Principle of dominance
3.Principle of segregation/Principle of purity of gametes 4. Principle of independent
assortment.
8. If hybridization is made between contrasting traits of a character the F 1 generation shows
only one trait and is called dominant and one trait that cannot express it is known as
recessive.
9. Codominance is the genetic phenomenon in which both of the alleles of a gene express
themselves in heterozygous condition.
10. When the phenotype of F1 heterozygous genotype lies between the phenotypes of parental
homozygous genotypes, it is called incomplete dominance.
11. The Law of Segregation states that every individual organism contains two alleles for each
trait, and that these alleles segregate (separate) during meiosis such that each gamete contains
only one of the alleles.
12. Mendel's law of independent assortment states that the alleles of two (or more) different
genes assort independently of one another during the course of inheritance.
13. An allele is an alternative form of a given gene.
14. Three or more kinds of genes which occupy the same locus are referred to as multiple alleles
and character governed by more than two allele called multiple allelism.
15. Organisms can be homozygous or heterozygous for a gene.
16. Homozygous means that the organism has two copies of the same allele for a gene and
heterozygous organism has two copies of the different allele for a gene.
17. Genotype is genetic constitution of an organism i.e., full hereditary information. Phenotype
is an organism's actual observed properties means external appearance
18. Epistasis (Greek=standing upon) is the masking genetic phenomenon when the phenotypic
effect of alleles at one gene masks or inhibits the expression of alleles of another gene.
19. The term “epistatis” was first of all used by Bateson (1909).

20. When F1 individuals are crossed with one of the two parents from which they have been
derived, then such a cross is called back cross.
21. Back cross can be of two types: Out cross and test cross.
22. When Tt (F1) is crossed with TT (Homozygous dominant parental), it is called out cross.
23. When Tt (F1) is crossed with tt (Homozygous recessive parental), it is called test cross.
24. A test cross is a way to explore the genotpye of F1 individuals.
25. Non-allelic or inter-allelic interactions also occur where the development of single character
is due to two or more genes affecting the expression of each other in various ways called
gene interaction.
26. The two or more genes interact in several manners some common interactions are as follows:
(1) Dominant epistasis (12:3:1)
(2) Recessive epistasis or supplementary gene interaction (9:3:4)
(3) Double recessive/complementary gene interaction (9:7)
(4) Inhibitory gene interaction/dominant recessive epistasis (13:3)
(5) Polymorphic gene interaction (9:6:1)
(6) Duplicate gene interaction/double dominant epistasis (15:1)
(7) Collaborative supplementary/modified gene interaction (9:3:3:1)
(8) Polymeric gene interaction (9:6:1)
6.5 GLOSSARY
Allele: Alternative form of a gene is called allele.
Back Cross: Backcross is a cross of F1 hybrid with one of its parents.
Dihybrid cross: A dihybrid cross describes a mating experiment between two organisms that
are identically hybrid for two traits.
Dominance: An allele or a gene that is expressed in an organism's phenotype, masking the effect
of the recessive allele or gene when present.
Epistasis: Epistasis is the interaction between non allelic genes that influences a phenotype.
F1= The first filial generation of offspring.
F2= The second filial generation of offspring.
Gene: A unit of heredity which is transferred from a parent to offspring.
Genetics: The scientific study of heredity.
Genotype: Genetic constitution of an organism.

Hemizygous: A condition individual having only one of a given pair of genes.

Heredity: The phenomenon of passing of traits genetically from one generation to another.
Homologous: Having the same or allelic genes with genetic loci usually arranged in the same
order homologous chromosomes.
Hybrid: The offspring resulting from the cross between parents of different species.
Monohybrid: A monohybrid cross is a breeding experiment conducted between parents, which
differ in one specific trait only.
Offspring: Young born of organisms, produced through reproduction.
Out cross: A backcross of F1 offspring with dominant parent.
Phenotype: The physical or external appearance of an organism as a result of the interaction of
its genotype and the environment.
Progeny: A genetic descendant or offspring, Collective offspring progeny.
Pureline: A population having a particular feature that has unchanged through many
generations. The organisms are homozygous and are said to Pureline or true-breed.
Selfing (Syn= Inbreeding): The union of male and female gametes from same haploid, diploid,
or polyploid organism.
Test cross: A backcross of F1 offspring with recessive parent.
6.6 SELF ASSESSMENT QUESTION

1. Who is called father of genetics:
(a) Mendel (b) Bateson
(c) Punnet (d) Morgan
2. Experimental material of Mendel was:

(a) Pigeon Pea (b) Garden Pea
(c) Sweat Pea (d) None of above
3. ABO blood group type in humans is an example of:

(a) Dominance (b) Codominance

(c) Incomplete dominance (d) Overdominance
4. Typical Mendelian dihybrid ratio in F2 generation is:

(a) 9:3:4 (b) 12:3:1
(c) 9:3:3:1 (d) 15:1
5. Typical Mendelian dihybrid ratio in F2 generation is:

(a) 9:3:3:1 (b) 27:9:9:9:3:3:3:1
(c) 9:3:4 (d) 27:9:9:3:3:3:1
6. If hybridization is made between contrasting traits of a character the F1 generation shows only
one trait this genetic phenomenon is called.
(a) Epistasis (b) Dominance
(c) Test cross (d) Backcross
7. Alternative form of gene is called:

(a) Phenotype (b) Phenocopy
(c) Allele (d) Genocopy
8. When F1 individuals are crossed with one of the two parents from which they have been
derived, then such a cross is called.
(a) Back cross (b) Test Cross
(c) Out cross (d) All of the above
9. An organism hving two copies of the same allele for a gene is called:
(a) Homozygous (b) Heterozygous
(c) Hemizygous (d) Hybrid
10. When F1 individuals are crossed with recessive parents then such a cross is called.:
(a) Out cross (b) Reciprocal Cross
(c) Dihybrid cross (d) Test Cross

11. The polymeric gene interaction modifies the typical 9:3:3:1 Mendelian F2 ratio into:
(a) 9:6:1 (b) 9:3:4
(c) 15:1 (d) 12:3:1
12. A genetic phenomenon of non allelic gene interaction in which a dominant gene or a
dominant gene pair inhibits or masks the expression of another dominant gene or gene pair is
called
(a) Dominant epistasis (b) Recessive epistasis
(c) Dominance (d) Overdominance
13. The term “epistatis” was first of all used by:

(a) Mendal (b) Bateson
(c) Punnet (d) Kolreuter
14. Self sterility in tobacco is the example of genetic phenomenon.

(a) Epistasis (b) Multiple allelism
(c) Dominance (d) All
15. In case of Double recessive/complementary gene interaction the F2 ratio becomes:

(a) 12:3:1 (b) 9:6:1
(c) 9:7 (d) 15:1

(1) The term epistasis was given by _______________.
(2) _________________is the genetic phenomenon in which both of the alleles of a gene express
themselves in heterozygous condition.
(3) The interaction of genes at different loci that affect the same character is called __________.
(4) ________________ condition means that an organism has two different alleles of a gene.
(5) Thipolymeric gene interaction modifies the typical 9:3:3:1 Mendelian ratio in to _________.
(6) In _________________gene interaction the non-allelic dominant gene inhibits the expression
of the other non-allelic dominant gene.

(7) When the phenotype of F1 heterozygous genotype lies between the phenotypes of parental
homozygous genotypes, is called ______________dominance.
(8) When F1 individuals are crossed with one of the two parents from which they have been
derived, such a cross is called ______________cross.
(9) Alternative form of a gene is called________________________.
(10) When F1 individual is crossed with recessive parent, then such a cross is called
_______________cross.

(1) When F1 individuals are crossed with one of the two parents from which they have been
derived, then such a cross is called test cross.
(2) Mendal coined the term epistasis.
(3) In Epistasiss one allele inhibits the expression of another allele of same gene.
(4) Dominance is allelic while epistasis is non allelic genetic phenomenon.
(5) Self sterility gene is an example of multiple allelism.
(6) A backcross of F1 offspring with dominant parent is called test cross.
(7) Principle of paired factor was not followed by haploid and polyploidy organisms.
(8) Roan Coat colour in cattle is an example of codominance.
(9) A genetic phenomenon of non allelic gene interaction in which a gene in its homozygous
recessive state masks the expression of non allelic gene or gene pair is called recessive
epistasis.
(10) Genetic constitution of an organism is called phenotype.

(1) Define genetics.
(2) Who is called father of genetics?
(3) Who coined the term epistasis?
(4) Define allele.
(5) Define dominance.
(6) What is the typical trihybrid ratio in F2 generation Mendelian inheritance?
(7) What is recessive epistasis?

(8) Describe inheritance pattern of comb in fowl?

(9) What is the F2 ratio in case of supplementary gene interaction?
(10) Define test cross.
6.6.1 Answer key: 1-(a), 2-(b), 3-(b), 4-(c), 5-(b), 6-(b), 7-(c), 8-(a), 9-(a), 10-(d), 11-(a), 12-
(a), 13-(b), 14-(b), 15-(c).
6.6.2 Answer key: 1-Bateson(1909), 2- Codominance, 3- Epistasis, 4- Heterozygous 5- 9:6:1,
6- Inhibitory, 7-Incomplete, 8- Back, 9- Allele, 10- Test.
10-False.
6.7 REFERENCES
 Bateson, W. 1909. Heredity and variation in modern lights. In Darwin and modern science
(eds. A. C. Seward), 85-101. Cambridge University Press, Cambridge.
 Bateson, W. and Punnett, R.C. 1908. Experimental studics in the physiology of heredity.
Reports to the Evol. Comm. Roy. Soc. Rpt. 4, Poultry, pp. 18-35.
 East, E.M. and Yarnell, S.H. 1929. Studies on self-sterility. VIII. Self-sterility allelomorphs.
Genetics 14:455-487.
 Kölreuter, J.G. (1761-1766), Vorläufige Nachricht von inigen das Geschlecht der Pflanzen
betreffenden Versuchen und Beobachtungen, nebst Fortsetzungen 1, 2 und 3, Leipzig: in der
Gleditschischen Handlung.
 Mendel, G. 1866. Versuche über Plflanzenhybriden. Verhand-lungen des naturforschenden
Vereines in Brünn, Bd. IV für das Jahr 1865, Abhandlungen, 3–47.
(http://www.esp.org/foundations/genetics/classical/gm-65.pdf ).
 Punnett, R. C. 1923 Heredity in poultry. London: Macmillan and Co.

 Brooker, R. 2015. Genetics: Analysis and Principles (5th Eds.). McGraw-Hill Publishing
Company.
 Gardner, E.J., Simmons, M.J. and Snustad, D.P. 2006. Principles of Genetics (8 th Ed.).
Wiley.

 Klug, W.S., Cummings, M.R., Spencer C. A., Palladino, M.A., Killian, D.2019. Concepts of
Genetics (12th Ed.). Pearson.
 Klug, W.S., Cummings, M.R., Spencer C. A., Palladino.2019. Concepts of Genetics (10 th
Ed.). Pearson.
 Pierce, B. A. 2017.Genetics: A Conceptual Approach (6th Ed.). W.H. Freeman.
 Singh, B.D. 2014. Fundamentals of Genetics. Kalyani Publshers, India.
 Singh, B.D. 2016. Genetics (2nd Ed.). Kalyani Publishers, India.

1. What do you understand by Epistasis?
2. Define dominant epistais with suitable example(s).
3. Differentiate between epistasis and dominance.
4. What is the recessive epistsis, define with example?
5. Define monohybrid and dihybrid cross and what is the typical Mendelian mono- and dihybrid
cross ratio in F2 generation?
6. Describe collaborative gene interaction with sitable examples.
7. Define backcross, testcross and out cross.
8. Define co-dominance with suitable examples.
9. What do you understand by gene interaction describe in brief.
10 Describe complementary gene interaction with example.

1. Bateson and Punnett performed an experiment on sweat pea and concluded that when two
white flowered varieties were crossed, F 1 hybrids were all purple coloured but in F 2 generation it
segregated in to purple and white colour. Give the parental genotype and describe the type of
gene interaction.
2. In a genetic experiment when purple pigmented paddy plant was crossed with green
pigmented plant, the F1 appeared green pigmented. In F2 the segregation of two types of
pigmentation was as: purple pigment 90, green Pigment 410.What may be the genetic basis for
the segregation of pigmentation in F2. Describe the type of gene interaction in this experiment

3. In summer squash white (W) colour is epistatic to that of an yellow (Y) which is dominant
over green colour gene. Find out the fruit colour progeny if following crosses performed and
describe the type of gene interaction.
1- WwYy x Wwyy and 2- wwYY x Wwyy.
4. In Fowl dominant gene R gives rose comb and the dominant gene P gives pea comb. When P
and R are present together gives walnut. The homozygous recessives of P and R produce single
comb. What will be the comb type in the following crosses and describe the type of gene
interactin in this case:-
A. RrPp x RrPp
B. rrPP x RrPp
C. Rrpp x RrPp
D. rrPp x RRPp
E. rrpp x Rrpp
5. Define non-Mendelian inheritance. Describe about different kinds of gene interaction with
suitable examples.
UNIT-7-IDENTIFICATION OF INDIAN VARIETIES OF

IMPORTANT CROPS

7.1 Objectives
7.2 Introduction
7.3 Identification of Indian varieties of important crop
7.3.1 Techniques used in identification of Indian varieties of important crops:
7.3.1.1 Karyotyping
7.3.1.2 Chromosome banding
7.3.1.3 Fluorescent in situ hybridization (FISH)
7.3.1.4 Comparative genomic hybridization
7.3.2 Crop Variety
7.3.2.1 Rice
7.3.2.2 Wheat
7.3.2.3 Cotton
7.3.2.4 Sugarcane
7.3.2.5 Tea
7.3.2.6 Coffee
7.4 Summary
7.5 Glossary
7.7 References
7.1 OBJECTIVES

To identify the Indian varieties of important crops.
7.2 INTRODUCTION
India produces many crops in the world and most of its population is relied on the crop and
agriculture industry. On the basis of seasons, crops are divided into Rabi, Kharif and Zaid.
Kharif crops are sown in June-July during monsoon period and harvested in September-october.
rice, jowar, bajra, maize, cotton, groundnut, jute, sugercane, turmeric and pulses are the common
examples. They need abundant water and hot weather to grow. Rabi crops are sown in October-
November and harvested in April-May. Wheat, oat, gram, pea, barley, potato, tomato, onion, oil
seeds (mustard, sunflower, sesame, and rapeseed), etc., are some examples. These crops need
warm weather to germinate and mature seed but cold weather to grow. Zaid crops are grown in
between March-June. They are also called early maturing crops. Cucumbers, Bitter Gourd,
Pumpkim, Watermelon, muskmelon, Moong Dal, etc., are some examples.
Also, major crops in India are divided into 4 another categories naming, Food crops which
include rice, what, maize, millets and pulses, etc; Cash crops including sugarcane, tobacco,
cotton, jute, oilseeds, etc; Plantation crops which has coffee, coconut, tea, rubber, etc; and
horticulture crops which contains fruits and vegetables.
Many varieties of these crops have been developed in India by various methods like Plant
Breeding. Plant Breeding is a discipline of scientific principle of plant sciences, genetics and
cytogenetic. Plant breeding aims to develop genetically superior plants by using and
understanding genetics in terms of economic importance for the mankind.
7.3. Identification of Indian Varieties of Important Crops
7.3.1. Technique used in identification of Indian varieties of important crops:
Due to advancement in the field of genetics and plant breeding, there are many approaches to
develop new varieties of plants. These varieties belong to the same species and hence do share
many morphological and phenotypic similarities. In order to distinguish them from one another,
reliable method of identification is needed. Karyotyping, chromosome banding, fluorescent in
situ hybridization (FISH) and comparative genomic hybridization (CGH) are such techniques.
7.3.1.1. Karyotyping (Fig 7.1)
1. It is the process which involves standard procedures of staining which reveals
chromosome characteristic features.

2. It is the process of pairing and chromosome ordering.

3. It can detect coarse genetic changes or changes in chromosome numbers.
4. It is based on the principle of enzymatic digestion of young leaf tissues i.e., shoot tips.
5. The process starts with short term culturing of the specimen derived cells.
6. After sufficient growth and multiplication of the culture, cells are arrested in metaphase
by adding colchicines.
7. The colchicine is used in poisoning the mitotic spindles.
8. In the metaphase, chromosomes are evenly and well-distributed which allows accurate
counting.
9. Next, cells are treated by hypotonic solution which causes the swelling of nuclei and
bursting of the cell.
10. A chemical fixative is added to nuclei and it is then treated with the various stains which
reveal structural features of the chromosomes.
11. Karyotypic differentiation is more prominent in the case of root tips than the shoot tips
because of longer and less condensed chromosomes in the root tip.
12. It reveals each chromosome as a specifc, unique and constant pattern of alternating dark
and light banding regions.
13. While studying the somatic cells of the Tritium erectum with the cold treatment method,
some chromosomes regions revealed the unique patterns of thin and less intensely stained
when compared to the rest of the chromosomes.

Fig.7.1: The process of Karyotyping in plants (https://www.slideshare.net/shruthibell iappa155/

ppt-on-karyotyping-chromosome-banding-and-chromosome-painting).
7.3.1.2. Chromosome Banding
Chromosome banding is referring to the pattern observed comprising of light and dark stain on
staining. The bands distinguish various locations in the chromosome. The dark and light bands
refer to the adjacent segments present in the chromosome which helps in genomic study and
evaluation.
Uses
1. To identify different regions in chromosomes.
2. To differentiate between euchromatin and heterochromatin.
3. To identify abnormal chromosome.
4. To understand various clinical features of chromosomes.
5. To identify different genes and their function.
6. To understand the molecular basis of any genetic disease.
7. To devise strategy to stabilize the unnatural changes in chromosome.

Types of Banding
(i) Q Banding (Fig 7.2 & 7.3)
1. The fluorescent dyes like quinacrine and quinacrine mustard bind preferentially to some
certain mitotic chromosomes region.
2. These fluorescent dyes interact with AT base pairs and hence staining is more in the case
of AT rich regions.
3. AT rich regions appear as bright bands called the Q bands.
4. These bands help in identifying all the chromosomes and their homologues in most of the
species.
5. It does not require any pre-treatment and is the simplest banding method.
6. This staining method can be used in Cricetulus griseus, Vicia faba and Triticum erectum.
7. It reveals the unique patterns of brightly fluorescent (light) regions alternate to non-
fluorescent (dark) regions which were produced in each chromosome.
8. Although, fluorescent bands are not permanent, and for visualization, ultra violet light is
needed.
9. This method does stain the chromosome ends and hence its use is considered to limited
extent.
10. Q banding is commonly used in Triticum, Scilla, Allium, Crepis, Lilium, Secale and
Vicia.

Fig.7.2: Q banding patterns in plant chromosome (Dematteis et al., 2006).
Fig.7.3: Q banding technique in plant chromosome (https://www.chegg.com

/learn/biology/introduction -to-biology/banding-pattern).

(ii) G Banding (Gustav Giemsa) (Fig 7.4)

1. Thus technique also reveals each chromosome segment and a unique pattern of bands by
the chromosome.
2. The Giemsa stain is used along with trypsin, urease as protease for staining and it yields
the banding pattern of normal mitotic chromosomes.
3. The dark regions formed are similar to Q bands and are called G bands.
4. The light regions are similar to the non-fluorescent dark bands which rose by the use of
fluorescent dyes.
5. A large number of bands of chromosome are produced in prophase and pro-metaphase.
6. The prophase and metaphase chromosomes contain a basic chromomeric structure which
is enhanced due to which G bands are produced.
7. This enhancement is caused by inducing some rearrangement of fibers away from the
light bands toward the G bands.
8. In plants, there are few species present which G bands are generated like Tulipa
gesneriana, Pinus resinosa and Vicia hajsatana.
9. Tribe like Triticeae fails to produce G bands in the chromosome due to the increased
condensation of the plant chromosomes.
10. Improper pre-treatment of plant chromosomes cause alteration in the organization of their
chemical constituents which make unresponsive to the procedure of G banding.

Fig.7.4: G banding chromosome patterns in plants (Chen et al., 1994).

(iii) R Banding
1. R stands for Reverse in R banding.
2. This technique was developed by Dutrillaux and Lejeune in the year 1971.
3. A reverse pattern from the G and Q banding methods is produced by mild denaturation
by heat and subsequent staining of chromosomes with Giemsa or a fluorochrome.
4. R bands are produced by GC specific fluorochromes.
5. Dark R bands are produced if chromosomes are stained by Giemsa.
6. The dark R bands produced are similar to light bands produced by G banding technique.
7. When fluorochrome dye like acridine orange or olivomycin is used, bands produced are
the reverse of Q banding technique. The R bands are of fluoresce bright green and the
faint red color indicates non-R bands.
8. This method is used in detecting structural rearrangements which involve ends of
chromosomes or Telomeres stained as T bands.
9. R bands are produced by some plant species only such as Scilla siberica, Vicia faba,
Alliums pp.

10. The R banding is produced when DNA and proteins in the G and R bands are denatured
selectively under different pH conditions, salt concentrations and temperature.
(iv) C Banding (Fig 7.5)
1. The chromosomes are stained with Giemsa and Ba(OH)2 in the constitutive
heterochromatin regions.
2. These regions are called as C bands which are present proximal to the centromeres of all
the chromosomes.
3. Constitutive heterochromatin resemble with the satellite DNA, consisting of short, highly
repeated base pairs sequences in tandem repeats among one or more regions of nearly all
chromosomes of most of the species.
4. The location of C bands is at various sites and also next to centromeres of each
chromosomes and next to secondary chromosome constrictions
5. C banding technique is limited as it does not allow recognition of individual
chromosomes with accuracy and precision.
6. The C bands are present in many species of Aegilops, Agropyron, Elymus, Hordeum,
Secale, Triticum, etc.
7. Some plant species like Allium crinatum, Horden spp. and Agropygron elonga do not
reveal C bands next to the centromeres of their chromosomes.
8. Due to this individual chromosomes in the somatic cells can be identified by their C
banding patterns.
9. Through this technique, 21 chromosomes are identified in the genome of Chinese Spring
and Norim 61 of T. aestivum.
10. This method is used in detecting aneuploids translocation and other structural
rearrangements and precise physical mapping of genes in the chromosomes.
11. The A and B genomes of T. turgidum have been identified which are considered as
inconsistent.

Fig.7.5: C banding patterns in plant chromosome (Jellen, 2016).

(v) N Banding (Fig 7.6)
1. This technique was developed by Matsui and Sasaki in 1973.
2. It stains the NORs in the chromosomes in the mammalian species which are different
from the N bands revealed in plant species.
3. The presence of N bands in plant species has some examples such as Triticum and
Aegilops species.
4. 21 chromosomes of common wheat have been identified by N banding patterns.
5. Also, improved N banding protocol revealed the 16 of the 21 chromosomes of common
wheat which includes five in the A genome.
6. 14 chromosomes of Aegilops variabilis are identified by N banding patterns.
7. The chromosomes have also been identified in barley, rye, lentils and Elymus spp. using
this technique.
8. This method can identify various types of aneuploids, alien additions and substitution
lines and translocation and deletions.
9. Some N bands are unknown and hence cannot be detected by this method as in T.
aestivum.

10. Many N bands are known to occupy the same position as that of C bands and hence it can
be concluded that 2 classes of heterochromatin occur in wheat, rye and other species.
11. Some regions of heterochromatin stains positive for both C and N banding. These are
known as C+N bands.
12. C+N bands possess multiple copies of the (GAA)n (GAG)n sequences in DNA.
Fig.7.6: N banding patterns in Barley (Singh & Tsuchiya, 1982).

(vi) Analysis (Fig 7.7)
1. A unique banding pattern of chromosome is revealed by this method.
2. It is more reliable staining method for the identification of chromosomes.
3. Five major banding techniques naming, Q, G, R, C and N provide precise cytogenetic and
phylogenetic analysis.

Fig.7.7: Banded Chromosome Patterns of M. sativa (Bauchan & Hossain, 1997).

7.3.1.3. Fluorescent in situ hybridization (FISH) (Fig 7.8)
1. FISH based karyotyping is a powerful cytogenetic tool to study chromosome
organization, behavior and evolution.
2. A probe mixture of centromeric and sub-telomeric satellite repeats, 5 S rDNA and
chromosome specific BAC clones is used in this technique.
3. This probe mixture helps in distinguishing all pairs of chromosome like 11 chromosome
pairs of the common bean.
4. The gene pools can be used in distinctly identifying several wild relatives and landraces
of common bean and other Phaseolus species.
5. The genetic evolution of plant can be studied by this method e.g. genus Phaseolus.
6. The study in Phaseolus genus has revealed that the chromosomal distribution of the
centromeric and subtelomeric satellite repeats is stable in common beans but copy
number of the repeats was variable.
7. Such variation suggests rapid amplification/reduction of the repeats in specific genomic
regions.
8. Although, copy numbers of centromeric repeats can be largely reduced or diverged due to
changes in chromosomal distributions due to rapid evolution of centromeric repeats.
9. Due to similar problem, the varied distribution pattern of subtelomeric repeats is also
observed.

10. The satellites revealed in FISH-based karyotyping system are active, rapidly evolving,
forming genomic features unique to individual common bean accessions
and Phaseolus species.
11. Similarly, studies have been performed in maize, soybean, and Brassica species by using
repetitive DNA probes.
12. This method has proven to be very useful in distinguishing the individual chromosomes,
and to identify variation in chromosome structure and repeat distributions, both between
and within species.
Fig.7.2: The Procedure of Fluorescent in situ hybridization (FISH) (https://www.creative-

diagnostics.com/in-situ-hybridization-and-fluorescence-in-situ-hybridization.htm)
7.3.1.4. Comparative genomic hybridization (CGH) (Fig 7.9)

1. It is an efficient approach to study entire genomes for variations in DNA copy numbers.
2. Firstly, the total genomic DNA is isolated for the test and reference cell populations.
3. The differential labeling allows following the experimental protocol easily.
4. The DNA is hybridized to allow the binding of sequences at different locations to be
easily distinguished.
5. Number of genomes can be compared simultaneously if labels used are suitable.
6. The inclusion of unlabeled Cot-1 DNA in the reaction causes suppression of
hybridization of highly repetitive sequences.
7. The physical position is mapped on the chromosomes by using metaphase chromosomes
for representing the genome and the location of copy number variations between the test
and the reference genomic DNA.
8. DNA microarrays containing elements are directly mapped to the genome sequences.
9. There is a proportional relationship between the relative hybridization intensity of the test
and reference signals at a given location with the relative copy number of those
sequences in the test and reference genomes.
10. This method can be used to determine the actual copy number associated with a ratio
level.
11. Several techniques like ligation-mediated polymerase chain reaction (PCR), degenerate
primer PCR using one or several sets of primers and rolling circle amplification are used
to amplify the starting material of genomes.
12. For hybridization, cDNAs, selected PCR products and oligonucleotides made by array
CGH can be used.
13. The reduced complexity representations of the genome produced by PCR techniques are
employed by hybridization with total genomic DNA.
14. Computational analysis of the genome sequence design the array elements which are
complementary to the sequences contained in the representation.

Fig.7.9: Comparative genomic hybridization (CGH) (Dorritie et al., 2004).

7.3.2. Crop Varieties
India is known for its agriculture importance. Many crop varieties, especially after green
revolution, have been developed in India. India produces both for its citizens and for the world.
Crops like wheat, rice, sugarcane, tea, coffee, cotton, etc. are the important part of Indian
agriculture and they are also important from the economic point of view.
7.3.2.1. Rice Varieties
It is one of the most important crops grown in country and is grown throughout the year. It needs
atmospheric moisture and rainfall for the irrigation. The rice fields are known as paddy fields and
they are flooded by water up to 10-12 cm deep in early stages. It is a predominantly tropical,
Kharif crop.
Scientific Classification:
Kingdom: Plantae
Clade: Tracheophytes
Clade: Angiosperms

Clade: Monocots
Clade: Commelinids
Order: Poales
Family: Poaceae
Genus: Oryza
Habit and Habitat: The tropical climate is optimum for rice varieties to grow. They have the
self-supporting growth form which grows in freshwater habitat. The soil can be clay or loamy.
The optimum temperature is 24oC. It covers the one third area of whole India’s land. West
Bengal, Punjab and Uttar Pradesh are the top producer states of the India. Other states include
Tamil Nadu, Andhra Pradesh, Jharkhand, Bihar, Uttarakhand, Chhattisgarh, Odisha, Karnataka,
Assam, Maharashtra, Haryana, Gujarat, Madhya Pradesh, Kerala and Kashmir Valley.
Crop Varieties:
1. Inter racial hybridization between japonicas and indicas give rise to more than 700
hybrid combinations.
2. The indica varieties are known for local condition adaptations and tolerance towards
diseases and pests whereas japonicas are known for high yielding capacity and response.
3. ADT-27 is developed in Tamil Nadu in India.
4. Inter-racial hybridization between semi-dwarf Taiwanese types/derivatives led to the
development of Taichung (Native) – I.
5. The two famous varieties developed by this inter racial hybridization are Padma and Jaya.
6. These developed varieties have got high yield potential.
7. The total 123 + 51 varieties were released first from this hybridization.
8. These semi-dwarf varieties are superior as they are efficient grain producer than the tall
traditional varieties.
7.3.2.2. Wheat Varieties
It is a Rabi crop and is the 2nd largest crop produced by India in the whole world. T is used as
staple food in north and north western India. It is a winter crop as it needs low temperature to
grow. The states of Uttar Pradesh, Punjab and Haryana are the top producers of wheat in the
country.
Kingdom: Plantae

Clade: Angiosperms
Clade: Monocots
Clade: Commelinids
Order: Poales
Family: Poaceae
Subfamily: Pooideae
Supertribe: Triticodae
Tribe: Triticeae
Genus: Triticum
Habit and Habitat: The ideal temperature range for wheat to grow is 10-15 oC for sowing and
21-26oC for harvesting. The rainfall should be less than 100 cm but more than the 75 cm for
wheat to thrive. Well drained fertile loamy soil and clayey soil is best for wheat to grow. They
are mostly grown in plain areas. The most common habitats are fields, roadsides, areas along
railroads, area near grain elevators and open waste areas.
Crop Varieties:
1. Wheat breeding in India was achieved by systematic manner in 1905 at the Imperial
Agricultural Research Institute, Pusa, and was developed Pusa4, Pusa6 and Pusa 12.
2. The main Indian varieties of Wheat are VL-832, VL-804, HS-365, HS-240, HD2687,
WH-147, PBW-343, WH-896 (d), PDW-233 (d), UP-2338, PBW-502, Shresth (HD
2687), Aditya (HD 2781), HW-1085, NP-200 (di), HW-741.
3. Wheat varieties like PbC518, PbC591, C273, C281 and C286 were developed by pure
line selection in indigenous material at Lyallpur, Kanpur, Sabour, Powarkheda, Niphad
and Pune.
4. Rust resistant varieties like NP783 and NP784 which were resistant to brown rust; NP785
and NP786 which were resistant to all races of yellow rust; NP789 and NP790 which
were resistant to black rust were developed in 1935.
5. Also, NP809 resistant to all the three rusts and loose smut was developed.
6. C306 is a tall variety developed by CCSHAU, Hisar for rainfed cultivation in NWPZ
during early sixties is still chosen by consumers due to their chapatti quality and other
traits.

7.3.2.3. Cotton Varieties

It is a Kharif crop. This fiber crop is also known as ‘White gold’. It does not need enough water
except at maturity. India holds 3rd rank in worldwide production of cotton. It grows in tropical
and subtropical regions. Cotton requires modest rainfall and uniformly high temperature of 21oC
to 30oC. The best considered soil for growing cotton is the black soils of Deccan and Malwa
plateau, alluvial soils of the Satluj-Ganga plain, red and laterite soils of the peninsular region.
Gujarat, Maharashtra and Andhra Pradesh are the major cotton producing states.
Kingdom: Plantae
Clade: Angiosperms
Clade: Eudicots
Clade: Rosids
Order: Malvales
Family: Malvaceae
Subfamily: Malvoideae
Tribe: Gossypieae
Genus: Gossypium
Habit and Habitat: It grows in tropical and subtropical regions. Cotton requires modest rainfall
of 40-100 cm. The uniformly high temperature of 21 oC to 30oC is best for cotton to grow. The
best considered soil for cotton to grow is the black soils of Deccan and Malwa plateau, alluvial
soils of the Satluj-Ganga plain and red and laterite soils of the peninsular region.
Crop Varieties:
1. All four species of cotton are produced in India; Gossypium arboreum and G. herbaceum
(Asian cotton), G. barbadense (Egyptian cotton or Sea Island Cotton or Peruvian Cotton
or Tanguish Cotton or Quality Cotton) and G. hirsutum (American cotton or Upload
cotton).
2. G. arboreum and G. herbaceum are diploid (2n=26) and are indigenous to the old world.
3. G. hirsutum contributes to the 90 % of the hybrid cotton production in India.
4. G. barbadense and G. hirsutum are tetraploid (2n=52) and are considered as New World
Cottons.

5. 90 varieties of upland cotton, 3 of Egyptian cotton, 39 of diploid cottons and 43 hybrids

have been released for commercial cultivation in different states of India after the
inception of All India Coordinated Cotton Improvement Project (AICCIP) in April, 1967.
6. The different states in which cotton varieties released were Punjab, Haryana, Rajasthan
and Western U.P.
7. Ludhiana is the main centre and Faridkot is the sub-centre for variety improvement work
of cotton.
8. 18 varieties of upland cotton and six of arboreum cotton have been released in Punjab
naming, LH 900, LH 1556, F846 and F 1378 in G. hirsutum and G.27, LD 327 and LD
491 in G. arboreum.
9. ‘H 4’ cotton hybrid was the world’s first cotton hybrid developed in India by Dr. C T
Patel (Father of hybrid cotton).
10. It has high potential yield and wide adaptability among farmers in Gujarat, Andhra
Pradesh, Karnataka, Maharashtra and Madhya Pradesh.
11. The first interspecific hybrid between G. hirsutum and G. barbadense gave rise to the
development of cotton hybrid, “Varalaxmi”.
12. It was released in Karnataka, Tamil Nadu, Andhra Pradesh and Maharashtra.
13. Heterosis breeding is also used for developing hybrids in several tetraploid cottons and
few in diploid cottons.
7.3.2.4. Sungarcane Varieties
It is considered as cash crop and India is the second largest producer of sugarcane in the world.
Sugarcane needs 7-8 months long rainy season. In India, North India and South India are
involved in sugarcane production but their properties are different. In north India, sugarcane
produced are of sub-tropical varieties and so have low sugar content whereas in south India,
tropical varieties and coastal areas hence have high sugar content and high yield.
Kingdom: Plantae
Clade: Angiosperms
Clade: Monocots
Clade: Commelinids

Order: Poales
Family: Poaceae
Genus: Saccharum
Habit and Habitat: Sugarcane is the perennial grass which belongs to the poaceae family. They
grow in the subtropical and tropical regions. The volcanic soil, clayey loamy soil, black cotton
soil, red loamy soil, brown loamy soil and alluvial soils of rivers are the best for sugarcane to
grow. The rainfall should have range from 75-150 cm. the optimum temperature varies from 20-
26o C.
Crop Varieties:
1. CoS 767 sugarcane varieties is not as popular as it deteriorates fast due to increased
incidence of GSD and red rot.
2. Co 09022 (Karan-12) sugarcane variety is developed from the open pollinated fluffs of
CoLk 8102 (GC).
3. Co 09022 (Karan-12) has high sugar, good quality and free from diseases with low
insect-pest incidence.
4. Co 05009 is a mid-late maturing sugarcane variety which is developed by progeny of Co
8353 x Co 62198 in Sugarcane Breeding Institute Regional Centre, Karnal.
5. Co 06030 is a mid-late maturing sugarcane variety which is developed by crossing
between the CoC 671 x IG 91-1100.
6. CoC 671 has high sugar variety and the other parent IG 91-1100 is an intergeneric hybrid
between CoC 772 and Erianthus arundinaceus which helps in maintain high yield and
vigour.
7. Co 06027 is mid late maturing sugarcane variety which is developed by the hybridization
crossing of CoC 671 x IG 91-1100 and then selection.
8. Again due to the same parents as CoC 671, Co 06027 has high sugar, high yield and
vigour.
9. Co 09004 is another sugarcane variety which has high yield, high quality and is again
early maturing sugarcane.
10. This variety is developed by the segregating progenies of the cross CoC 671 x CoT 8201.
11. Parent CoC 671 has high sugar variety and act as female parent while CoT 8201 act as
male parent, is a high cane tonnage plant.

7.3.2.5. Tea Varieties

It is an evergreen plant. It grows in tropical and subtropical climates. It is not easy to cultivate tea
plant as it needs intensive care and laborers to look out. They do not prefer much light and hence
grow in shade. India has become 2nd major producer of the world and is 1st on rank as largest
consumer of the world.
Kingdom: Plantae
Clade: Angiosperms
Clade: Eudicots
Clade: Asterids
Order: Ericales
Family: Theaceae
Genus: Camellia
Habit and Habitat: Heavy rainfall of 150-250 cm is required by tree plant although, water
logging is not tolerable by roots of tea plant. It requires sloppy areas with well-drained forest
loam soil. Tea is grown in 16 states of India among which Assam, West Bengal, Tamil Nadu and
Kerala produce 95% of total tea production.
Crop Varieties:
1. Teas are generally diploid (2n=30) but triploid (2n=45) tea plants are also present.
2. The breeding give rise to natural diploids, triploids, tetraploids, aneuploids and
polyploidy varieties.
3. Most popular varieties of Tea plant used for commercial purposes are UPASI: BSS-1,
UPASI: BSS-2, UPASI: BSS-3 and UPASI: BSS-4.
4. These cultivars are produced by combinations of a common parent TRI 2025 and UPASI-
10, UPASI-2, UPASI-9 and UPASI-15.
5. The crossing of parents CR-6017 and UPASI-8 produces UPASI: BSS-5 tea plant hybrid.
6. TISS-1 variety is produced by breeding between UPASI-9 and TRI-2026.
7.3.2.6. Coffee Varieties
A coffee plantation can be found on the gentle to moderate mountainous slopes. They need hot
and humid climate to grow. They are generally grown under shady trees. Strong sun shine and

high temperature from 30°C, frost and snowfall, etc., are injurious for coffee cultivation.
Moreover, dry weather condition is optimum for plants at the time of ripening of berries. Coffee
plantation needs skilled labor.
Kingdom: Plantae
Clade: Angiosperms
Clade: Eudicots
Clade: Asterids
Order: Gentianales
Family: Rubiaceae
Tribe: Coffeeae
Genus: Coffea
Habit and Habitat: Coffee is generally grown in shade. The two tiers of shade are provided to
these plants. Required altitude range varies from 1,000 to 1,500 m above sea level for arabica
(premier coffee), and 500 to 1,000 m for robusta (lower quality). They need well-drained, rich
friable loamy soil with humus and mineral conditions, favors high and rich organic matter.
Rainfall ranges between the 150-250 cm. Karnataka, Kerala and Tamil Nadu are the major coffee
producing states.
Crop Varieties:
1. Coffee arabica has much variability and hybrids in the form of dwarf, semi-dwarf, tall in
plant size; erect, spreading, drooping in branching habit; small, medium, bold in fruit
size; early, late in fruit ripening; in quality traits and yield potential.
2. Arabica coffee (Coffea arabica L) produces superior quality coffee but is susceptible to
diseases like leaf rust and pests like white stem borer.
3. 13 selections are produced from the Arabica coffee (Coffea arabica L) out of which 7 are
used for commercial purposes.
4. These selections have properties like leaf rust resistance, high yielding potential, wide
adaptability and superior quality
5. Standard breeding strategies like inter-varietal hybridization followed by pedigree
selection in Sln.2 and Sln.3 produces coffee hybrids.

6. The pure line selection in Sln.1, Sln.4 and Sln.8 produces the hybrids
7. Interspecific hybridization followed by backcross breeding in Sln.6, double crosses in
Sln.10, multiple crosses in Sln.7.3 and introgressive breeding in Sln.5A, Sln.5B, Sln. 9,
Sln.12 and Sln.13 using spontaneous tetraploid interspecific hybrids like Devamachy and
Hybrido De Timor (HDT), etc are used to develop coffee hybrids.
8. The S.288 (Sln.1) was the first selection developed by pure line selection from S.26,
which is a putative natural hybrid of C. arabica and C. liberica.
9. The S.288 plants are vigorous growth and manifested resistance to leaf rust race 1 and 2.
10. This selection line is superior to other varieties like ‘chiks’ and “kents’.
11. Fruits of this selection are round with broad disc and orange yellow to red in color,
popularly known as ‘golden drops’.
12. This selection is known for its general adaptability and is a moderate yielder (800 to 1000
kg ha-1) but produces high percentage of defective beans (20 to 30%). The liquor quality
is rated as Fair Average Quality.
13. Sln.2 (S.333) is a hybrid of S.31 and S.22 and has properties like hybrid vigor and
resistance to leaf rust.
14. Although, it does not gain much popularity due to the high bean abnormalities.
15. Sln.3 (S.795) is developed by crossing between S.288 and ‘kents’ coffee.
16. This selection is vigorous and wide spreading with profuse growth and a yield potential
of 1500 to 2000 kg ha-1.
17. They shows resistance towards leaf rust race 1 and 2 but are susceptible to leaf rust race
7,8,12 and 16.
18. Sln.4 arises by composite selection of three Ethiopian arabica collections which are
Cioccie, Agaro and Tafarikela.
19. Fruits of this selection are relatively small and beans are bluish green, long and bold with
65% ‘A’ grade and excellent liquor quality.
20. Cioccie and Agaro are resistant to leaf rust races I, II and VIII.
21. Tafarikela is susceptible to rust and exhibits horizontal resistance by showing very less
rust build up under field conditions.

22. Sln.5 selection line includes two separate families of cross bred lines involving
Devamachy and S.881 which is a wild arabica collection from Rume Sudan and
Devamachy and S.333.
23. Sln.11 is derived from interspecific hybridization between C. arabica and
C. eygenioides and spontaneously obtained by amphiploidy.
24. Sln.12 also called Cauvery is a semi-dwarf genotype which is derived from a cross
between caturra and HDT.
7.4- SUMMARY
The cytogenetics is a reliable method of studying plant chromosomes and genome. It provides
precise information about the physical structure of the chromosomes and location of genes. By
using genomics, genetics and cytogenetics; the success rate of plant breeding is increased. There
are number of varieties produced through this method which causes the development of better
quality, high yield and seed product from the hybrids. There are many methods to study the
chromosomal rearrangements, physical structure of chromosome and gene locations such as
Karyotyping, Chromosome banding, Fluorescent in situ hybridization (FISH) and Comparative
genomic hybridization (CGH). Various variety hybrids of wheat, rice, cotton, sugarcane, tea and
coffee are made within India which are used all over the world and has better properties from the
wild cultivars.
7.5- GLOSSARY
Accurate- It means correct in all details or exact.
Agriculture- It is the science or practice of farming which includes cultivation of crops and the
rearing of animals in order to provide food, wool, and other products.
Aneuploid- It refers to an abnormal number of chromosomes in a haploid set.
Centromeres- It is the region of a chromosome in which the microtubules of the spindle are
attached by the kinetochore during cell division.
Chromosome- It is a threadlike structure of nucleic acids and protein which is found in the
nucleus of most living cells and carry genetic information in the form of genes.
Constriction- It is the action of making something narrower through pressure or by becoming
narrower; tightening.
Denaturation- It is the method of breaking the weak linkages, or bonds of protein, nucleic acids,
etc.

Fluorochrome- It is a chemical which fluoresces in the presence of a label in biological

research.
Heterochromatin- It is the chromosome material with different density from normal which is
usually greater and in which the activity of the genes can be modified or suppressed.
Homologue- It refers to the similarity in the internal or chromosomal structures
Horticulture- It is the science and art of growing fruits, vegetables, flowers, or ornamental
plants.
Ligation- It is the process of joining of two molecules or DNA.
Metaphase- It refers to the second stage of cell division which occurs between prophase and
anaphase and in which the chromosomes become attached to the spindle fibers.
Mitosis- It is a process of cell division which results in two daughter cells, each having the same
number and kind of chromosomes with that of parent nucleus.
Monsoon- Also refer to as rainy season.
Morphology- It means external particular form, shape, or structure.
Phenotype- It is the set of observable characteristics of an individual from outside which results
from the interaction of its genotype with the environment.
Plantation- It refers to a land on which crops such as coffee, sugar, and tobacco are grown.
Precision- It refers to the quality, condition, or fact of being exact and accurate.
Prophase- It is the first stage of cell division which occurs before metaphase. The chromosomes
are visible as paired chromatids and the nuclear envelope disappears. The first prophase of
meiosis includes the reduction division.
Telomeres- It is a region of repetitive nucleotide sequences at each end of a chromosome in
which the nucleotide is protected by the end of the chromosome from deterioration or from
fusion with neighboring chromosomes.
Translocation- It is the movement of something from one place to another.
7.6- SELF ASSESSMENT QUESTIONS

1. Co 09022 also called ________, has high sugar, good quality and free from diseases with
low insect-pest incidence.
2. ___________ coffee is susceptible to rust.
3. Inter racial hybridization is the process which involves standard procedures of staining
which reveals __________ characteristic features.

4. Sln.12 also called __________ is derived from a cross between caturra and HDT.
5. ___________ is a mid-late maturing sugarcane variety which is developed by crossing
between the CoC 671 x IG 91-1100.
6. Sln.11 is derived from ___________ between C. Arabica and C. eygenioides.
7. The japonicas and indicas give rise to more than 700 hybrid combinations by
____________.
8. AT rich regions appear as bright bands called the _______________.
9. The ____________ produced are similar to light bands produced by G banding
technique.
10. The fluorochrome dye _______ or _________ is used to produce bands which are the
reverse of Q banding technique.
11. The ________ provides the bright green fluorescence.
12. The chromosomes are stained with _________ in the constitutive heterochromatin
regions.
13. Unknown ___________ are detected by banding technique in T. aestivum.
14. ___________ contain the elements which are directly mapped to the genome sequences.
ANSWER TO SELF ASSESSMENT QUESTIONS

1. Karan-12
2. Tafarikela
3. Chromosome
4. Cauvery
5. Co 06030
6. Interspecific hybridization
7. Inter racial hybridization
8. Q bands
9. Dark R bands
10. Acridine orange, Olivomycin
11. R bands
12. Giemsa
13. N bands
14. DNA microarrays

7.7- REFERENCES
1. Anamthawat-Jónsson, K. (2004). Preparation of chromosomes from plant leaf meristems
for karyotype analysis and in situ hybridization. Methods in Cell Science, 25(3-4), 91-95.
2. Banding Pattern. https://www.chegg.com/learn/biology/introduction-to-biology/banding-
pattern
3. Bauchan, G. R., & Hossain, M. A. (1997). Karyotypic analysis of C-banded
chromosomes of diploid alfalfa: Medicago sativa ssp. caerulea and ssp. falcata and their
hybrid. Journal of Heredity, 88(6), 533-537.
4. Cheema, A. K. (2018). Plant breeding its applications and future prospects. Int. J. Eng.
Technol. Sci. Res, 5, 88-94.
5. Chen, R., Song, W., Li, X., & An, Z. (1994). Chromosome G-banding in plants by
inducing with trypsin and urea. Cell Research, 4(1), 79-87.
6. Chromosome Banding and Karyotyping Ppt. (2020) Kromosom Saints.
https://kromosomsaints.blogspot.com/2020/12/chromosome-banding-and-karyotyping-
ppt.html
7. Das, S. C., Das, S., & Hazarika, M. (2012). Breeding of the tea plant (Camellia sinensis)
in India. In Global Tea Breeding (pp. 69-124). Springer, Berlin, Heidelberg.
8. Dematteis, M., Fernández, A., & Acosta, A. D. (2006). Heterochromatin variation in
Oziroë argentinensis (Hyacinthaceae) revealed by florescent banding. Caryologia, 59(2),
104-111.
9. Dorritie, K., Montagna, C., Difilippantonio, M. J., & Ried, T. (2004). Advanced
molecular cytogenetics in human and mouse. Expert review of molecular
diagnostics, 4(5), 663-676.
10. Fedak, G., & Kim, N. S. (2008). Tools and methodologies for cytogenetic studies of plant
chromosomes. Cytology and Genetics, 42(3), 189-203.
11. Gupta, A., Singh, C., Kumar, V., Tyagi, B. S., Tiwari, V., Chatrath, R., & Singh, G. P.
(2018). Wheat varieties notified in India since 1965. ICAR-Indian Institute of Wheat &
Barley Research, Karnal-132001, India.
12. Iwata-Otsubo, A., Radke, B., Findley, S., Abernathy, B., Vallejos, C. E., & Jackson, S. A.
(2016). Fluorescence in situ hybridization (FISH)-based karyotyping reveals rapid

evolution of centromeric and subtelomeric repeats in common bean (Phaseolus vulgaris)

and relatives. G3: Genes, Genomes, Genetics, 6(4), 1013-1022.
13. Jellen, E. N. (2016). C-banding of plant chromosomes. In Plant cytogenetics (pp. 1-5).
Humana Press, New York, NY.
14. Pinkel, D., & Albertson, D. G. (2005). Comparative genomic hybridization. Annu. Rev.
Genomics Hum. Genet., 6, 331-354.
15. Shakoori, A. R. (2017). Fluorescence in situ hybridization (FISH) and its applications.
In Chromosome Structure and Aberrations (pp. 343-367). Springer, New Delhi.
16. Shukla, S. K., Zubair, A., Awasthi, S. K., & Pathak, A. D. (2018). Sugarcane varieties
identified by AICRP (S) in India. ICAR-AICRP on Sugarcane. 1-132
17. Singh, P., & Kairon, M. S. (2001). Cotton varieties and hybrids. CICR Technical Bulletin
no: 13, 15.
18. Singh, R. J., & Tsuchiya, T. (1982). Identification and designation of telocentric
chromosomes in barley by means of Giemsa N-banding technique. Theoretical and
Applied Genetics, 64(1), 13-24.
19. https://www.creative-diagnostics.com/in-situ-hybridization-and-fluorescence-in-situ-
hybridization.htm
7.8- SUGGESTED READINGS

1. http://apeda.gov.in/apedawebsite/SubHead_Products/Wheat.htm#:~:text=Varieties
%3A,di)%2C%20HW%2D741.
2. http://drdpat.bih.nic.in/Rice%20Varieties%20in%20India.htm
3. http://www.businessworld.in/article/India-s-Cotton-Fact-Sheet-2017/21-09-2017-126710/
4. https://indianestates.co.in/development-of-indian-coffee-varieties/
5. https://www.gktoday.in/gk/major-crops-of-india/
6. https://www.patnauniversity.ac.in/e-content/science/botany/l/MScBot12.pdf
7.9- TERMINAL QUESTIONS

1. Describe the different techniques used for studying chromosomes.
2. What is the R Banding technique? Explain.
3. Provide a detail with different coffee varieties in India.
4. Explain the Triticum hybrid varieties and their breeding in detail.
5. Explain rice variety found in India.

6. Give a detail account of cotton variety in India.

BLOCK-3 PLANT DEVELOPMENT

UNIT-8-STUDY OF CYTOHISTOLOGICAL ZONES IN

THE SHOOT APICAL MERISTEM (SAM) IN
SECTIONED AND DOUBLE STAINED SLIDES
8.1-Objectives
8.2-Introduction
8.3- Study of Cytohistological zones in the shoot apical meristem (SAM) in sectioned and double
stained slides
8.4-Summary
8.5-Glossary
8.6-Self Assessment Questions
8.7-References
8.8-Suggested Readings
8.9-Terminal Questions
8.1 OBJECTIVES

 Understand the plant meristematic tissue.
 Understand the different types of meristematic tissues found in plants.
 Understand the sectioning and staining of meristematic tissue.
 Understand the cytohistological zones of shoot apical meristem.
8.2 INTRODUCTION
Meristem is a group of undifferentiated tissue found in plants has a capacity of division.
The term meristem was first coined by a Swiss botanist Carl Wilhelm von Nägeli in 1858. The term
meristem was mentioned in his book "Contributions to Scientific Botany". It is derived from the Greek
word 'merizein' which means 'to divide'. Meristems contain a population of cells with active cell
division which serves to constantly replenish the meristem and that differentiates into plant
organs and tissues. So a meristmatic tissue is a group of cells that are in continuous state of
division or retain their power of division. Meristematic cells have some characteristics like:
• They are generally round, oval or iso-diametric in shape.
• They have compact arrangement i.e. without intercellular spaces and with dense
cytoplasm.
• They contain large prominent nucleus with small vacuoles or without vacuoles.
• Cells have thin cell wall generally without secondary wall and don’t store food material
• The most striking feature is the capacity of division i.e., always in active state of division
and divide in a plane.
During postembryonic development, all plant organs are derived from a few meristematic cells
found in specialized structures and positions called apical meristems. Apical meristems are the
completely undifferentiated meristems, usually positioned at shoot apex and root apex. Apical
meristems are employed as synonyms for both root and shoot apical meristem. The shoot apical
meristem (SAM) is terminally positioned minute but complex structure that is covered by newly
developing lateral organs like leaves or bracts. According to Wardlaw (1957) and Cutter (1965)
the shoot apical meristem (SAM) comprises two groups of cells: the initial or source cells and

Fig.8.1: Meristematic Tissues in Plants (a) Stem Apex (b) Root Apex
the progenitor cells for tissues and lateral organs. The principal functions of the SAM (shoot
apical meristem) is the formation of new cells by division for the growth shoot axis and the
initial cells for lateral organs, such as branches, leaves, protective and accessory organs, etc.
The shoot apex is radially symmetrical. There occur great variations in shape and size of the
shoot apices among the spermatophytes. Usually the shoot apex in most of the plants is more or
less convex. In Anacharis, and some grasses it is a narrow cone with a rounded tip. According to
Gifford (1950) in some cases,e.g. in Hibiscus syriacus, Drimys, etc, it is slightly concave.
Clowes (1961) reported the size of Cycas revoluta shoot apex at the level of youngest leaf
primordium to be 3300 μm in width.
Classification of meristems
There are three types of meristems:
1. Apical meristems: The meristems present at the tips of roots and shoot are called apical
meristems. Apical meristems are involved in the extension of plant body, continuing for a long
period of time at the-apices of roots and stems. The cells derived from apical meristems are
grouped into three types (Fig.8.2):
• Protoderm: This is the surface layer . Its cells are differentiated into epidermal systems.
 Procambium: They are also called provascular tissues. Their cells form different parts of
vascular systems at ma urity.
 Ground meristem: These cells of these tissues form differ of ground tissues at maturity.

2. Intercalary meristems: The meristem situated at the bases of internodes is called

intercalary meristem. These are the parts of apical meristem, get separated from apex
meristem and play important role in the production of leaves and flower.
3. Lateral meristems:. Vascular and cork cambium are the example of lateral meristems. They
play an important role in the increase in diameter of stem and root. So they are involved
in secondary growth.
Fig.8.2: Positions of meristems

Organization of cells in Shoot Apical Meristem
There are two concepts about the organization of cells in the .apical meristems.
1. Histogen theory: This theory was put forward by Hanstein in 1870. According to this theory
the apical meristematic tissues arc divided into three Zones (Fig. 8.3).
(a) Dermatogens: This zone consist the cells of the surface layer. It gives rise to the epidermis.
(b) Periblem: This zone comprise; the cells of the outer region lying under the surface layer.
They give rise to the cortex or outer portion of the stem.
(c) Plerome: This zone comprises the central mass of cells. It gives rise to the vascular system
and the pith.
2. Tunica-Corpus theory This theory was given by Schmidt in 1924. According to this
concept, the dividing cells in the apical meristem are arranged in two zones (Fig.8.4):

Fig.8.3: Three Histogenic Zones in SAM

Tunica: The outer zone is called tunica composed composed of one or two layers of superficial
cells. The epidermis arises from the outer layer of tunica. Single layered tunica is present in
monocot plants. Dicots have two layered tunica.
Corpus: The central zone is called corpus which is covered by tunica cells. The cells of corpus
divide in all planes. Inner tissues like cortex and vascular tissues are derived from both tunica
and corpus.
Fig.8.4: Apical Meristem Organization In Tunica And Corpus

3. Histogenic layer theory: This theory “as put forward by Dermen in 1868. He proposed
the concept of primary histogenic layers. According to him. there are three basic
histogenic layers in angiosperms which named them as L-I-L-II.L-III gives rise to
vascular tissues and pith region (Fig.8.5).

Fig.8.5: Histogenic Layers in Shoot Apex

4. Cytohistological zonation theory: This theory was proposed by Foster in 1939. In
gymnosperms tunica like layers not found which led Foster to divide shoot apex
arganization of Ginkgo biloba on the basis of planes of divisions, size of cell and nucleus,
differential staining and orientation of cell division. He recognized four interrelated
zones.
(i) Apical Initial group
(ii) Central Mother Cell Zone
(iii) Rib Meristem
(iv)Flank orperipheral meristem ( Fig.8.6)
Fig.8.6: L.S. of Shoot meristem of Ginkgo biloba showing schematic representation of

cytohistological zonation
8.3- STUDY OF CYTOHISTOLOGICAL ZONES IN THE SHOOT

APICAL MERISTEM (SAM) IN SECTIONED AND DOUBLE
STAINED SLIDES
The cytohistological zones in SAM can be studied by maceration techniques applied with
suitable stains. The technique of maceration including the nature of the chemicals used depends
mainly on the hardness of the tissues to be macerated. On the nature of tissues Jeffrey’s Method
or Quick methods can be used in maceration. Jeffrey’s method, although quite adequate for all
kinds of tissues, is a very slow method. Quick method is suitable for herbaceous material like
Cucurbita. In former method the material (dry or fresh) is cut into small slices about 0.5 mm
thick. Boil and cool repeatedly until it becomes free of air. Macerate in a solution of equal parts
of 10% nitric acid and 10% chromic acid. The material with the solution may be heated in a
paraffin bath for woody tissues but not for soft and herbaceous tissues. The material is now
stained with any suitable stain, such as eosin or water soluble safranin, mounted in 50%
glycerine and observed. For permanent staining, leave the macerated tissue in 1% safranin for 6
hours, rinse thoroughly in water and then dehydrate by rapid addition of hygrobutol. Give two
changes of pure hygrobutol and then add a little balsam highly diluted with hygrobutol.
Evaporate it down to a mounting consistency and mount on slides. Herbaceous materials, such as
Cucurbita stems are sliced into thin pieces and taken in a test tube along with a little 10% or 20%
KOH solution, more KOH solution is added, if necessary. When the tissue comes out in shreds, it
is washed and stained as before. Siliques (for embryos) or seedlings are fixed in 3%
glutaraldehyde, 1.5% acrolein, 1.6% paraformaldehyde and postfixed in 1% osmium tetroxide.
Fixed material is dehydrated through an ethanol series and embedded in Spurr’s medium. 1 mm
sections are cut and stained with 0.5% methylene blue in 0.5% borate. The purple red or blue
stained samples are observed under microscope. On observation these traits in root apical
meristem and the shoot apical meristem would be seen .The general features of meristematic
mells are (a) isodiametric shape(b) thin primary cell wall (c) dense cytoplasm with few tiny
vacuoles (e) relatively large nucleus (f) frequent cell divisions (fig. 8.7)

Fig. 8.7: Densely stained cells of: (A) Shoot apical meristem (B) The root apical meristem, (C)
Cells from the center of the RAM: dense cytoplasm with clearly visible nuclei (D)
Enlarged view of (C).
Slice the material Cut herbaceous material in thin

(SAM/RAM) in small pieces pieces
(0.5mm)
Boil in test tube Add 10-20% KOH solution in

test tube
Cool down Boil with gentle shaking
Macerate in 10% nitric Add 10% nitric acid + 10%

acid + 10% chromic acid chromic acid
Stain eith eosin or Wash and stain

safranin (left for 6hrs)
Observe under Observe under microscope

microscope
Fig. 8.8:
Flow chart of (A) Jeffrey’s method (B) Quick Method
The shoot apical meristem generates stem, leaves, and lateral shoot meristems during the entire
shoot ontogeny. Vegetative leaves are generated by the meristem in the vegetative
developmental phase, while in the reproductive phase either bracts subtending lateral flower
primordia or perianth and strictly reproductive organs are formed. Meristem growth is fully
characterized by the principal growth rates, directions, volumetric, and aerial growth rates.
Growth modelling or sequential in vivo methods of meristem observation complemented by
growth quantification allow the above growth variables to be estimated. Indirectly, growth is

assessed by cell division rates and other cell cycle parameters. Temporal and spatial changes of
growth and geometry take place at the meristem during the transition from the vegetative to the
reproductive phase. During the vegetative phase, meristem growth is generally indeterminate. In
the reproductive phase it is almost always determinate, but the extent of determinacy depends on
the inflorescence architecture. In the vegetative phase the central meristem zone is the slowest
growing region. The transition from the vegetative to the reproductive phase is accompanied by
an increase in mitotic activity in this zone. The more determinate is the meristem growth, the
stronger is this mitotic activation. However, regardless of the extent of the activation, in
angiosperms the tunica/corpus structure of the meristem is preserved and therefore the mitotic
activity of germ line cells remains relatively low. In the case of the thoroughly studied model
angiosperm plant Arabidopsis thaliana, it is important to recognize that the flower primordium
develops in the axil of a rudimentary bract. Another important feature of growth of the
inflorescence shoot apical meristem is the heterogeneity of the peripheral zone. Finally, the role
of mechanical factors in growth and functioning of the meristem needs further investigation.
Shoot apex has apical meristems. The apical meristems of the shoot is more complex than the
root. Shoot apex has two types of arrangements:
(a) In lower vascular plants the apical meristem of the shoot is more complex than the
shoot apical meristem. The activity of the shoot apcial meristem increases the length of the plant
body and also forms leaves and lateral branches (fig.8.9)
(A) (B)

Fig.8.9: Shoot Apical Meristem (SAM) in (A) Selaginella and (B) Equistem Shoot
(b) In higher vascular plants i.e. gymnosperms and angiosperms the shoot apex consists
of a set of meristematic cells. These cells differentiate into different zones on the basis of their
meristematic activity (fig.8.10).
(A) (B)
Fig.8.10: Shoot Apical Meristem (SAM) in Higher Vascular Plants
8.4 SUMMARY
1. Meristem is a group of undifferentiated tissue found in plants has a capacity of division.
2. The term meristem was first coined by a Swiss botanist Carl Wilhelm von Nägeli in 1858.
3. Meristem is the tissues which the cells are undifferentiated and has the capacity of
division.
4. The term meristem has been derived from Greek word meristos, means divisible.
5. Meristems contain a population of cells with active cell division which serves to
constantly replenish the meristem and that differentiate into plant organs and tissues.
6. Initially all embryonic cells of an embryo have the capacity to divide and multiply but as
the embryo develops into a plant body.
7. During postembryonic development, all plant organs are derived from a few meristematic
cells found in specialized structures and positions called apical meristems.
8. Apical meristems are the completely undifferentiated meristems, usually positioned at
shoot apex and root apex. A
9. Apical meristems are employed as synonyms for both root and shoot apical meristem.

10. The shoot apical meristem (SAM) is terminally positioned minute but complex structure
that is covered by newly developing lateral organs like leaves or bracts.
11. The meristematic activity provides growth and healing in plants.
12. Before the occurrence of any cell division, usually cells become enlarged accompanied
with addition of protoplasmic and cell wall material.
13. They are generally round, oval or isodiametric in shape.
14. During postembryonic development, all plant organs derived from a few meristematic
cells found in specialized structures and positions are called apical meristems.
15. Apical meristems are the completely undifferentiated meristems, usually positioned at
shoot apex and root apex.
16. Apical meristems are employed as synonyms for both shoot and root apical meristem.
17. The shoot apical meristem (SAM) terminally positioned minute but complex structure
that covered by newly developing lateral organs like leaves or bracts.
18. There are three types of meristems: apical meristems, intercalary meristems, lateral
meristems.
19. The principal functions of the SAM (shoot apical meristem) is the formation of new cells
by division for the growth shoot axis and the initial cells for lateral organs, such as
branches, leaves, protective and accessory organs etc.
8.5 GLOSSARY
Anticlinal: occurring at right angles to the surface or circumference of a plant organ.

Corpus: The central zone is called corpus which is covered by tunica cells.
Cytohistological: the integrated study of cells and tissues.
Dermatogens: the outer primary meristem of a plant which gives rise to epidermis.
Development: the progressive changes in size, shape, and function during the life of an
organism.
Dicot: member of the flowering plants, or angiosperms that has a pair of leaves, or cotyledons, in
the embryo of the seed.
Embryo: An embryo is the early stage of development of a multicellular organism.

Epidermis: The epidermis is a single layer of cells that covers the leaves, flowers, roots and
stems of plants.
Histogen: a zone or clearly delimited region of primary tissue in plants from which the specific
organ are produced.
Internodes: internodes are those intervals between the nodes.
Isodiametric: a cell having a similar diameter in all planes.
Meristem: region of cells capable of division and growth in plants.
Meristos: Greek term used in relation to meristem is 'meristos' which means 'divisible'.
Nodes: nodes are those critical areas from which leaves, branches, and aerial roots grow out
from the stem.
Periblem: a primary meristem that gives rise to the cortex and is located between plerome and
dermatogens.
Periblem: a primary meristem that gives rise to the cortex and is located between plerome and
dermatogens.
Periclinal: Periclinal cell divisions are the ones that occur parallel to the tissue or organ surface.
Plerome: the central core of primary meristem of a plant or plant part that according to the
histogen theory gives rise to the stele.
Primordium: an organ, structure, or tissue in the earliest stage of development.
Protoderm: the outer primary meristem of a plant or plant part.
Root apex: the growing tip of the plant shoot.
Shoot apical meristem (SAM): the region in the growing shoot containing meristematic cells.
Spurr’s medium: low viscosity embedding medium, introduced in 1969 and used vinyl
cyclohexene dioxide (VCD or ERL 4206) as the low viscosity epoxy resin together.
Tissue: A tissue is a group of cells, in close proximity, organized to perform one or more
specific functions.
Tunica: the outer layer of cells, which covers tunica.
Vascular: plants with specialized vascular tissue.

1. Meristem is:

(a) Mature cells (b) Undifferentiated mass of cells

(c) Collenchyma cells (d)All of above
2. Apical meristem found in aerial part of plant tips:
(a)RAM (b) SAM
(c) Both (d) None of above
3. Growing part of plant tips composed of:
(a) Differentiated cells (b) Meristmetic cells
(c) Cambial cells (d) None of above
4. Tunica-Corpus theory given by:
(a) Lamark (b) Hanstein
(c) Buwat (d) Schmidt
5. Dermatogens give rise:
(a) Dermal layer (b) Cortex
(c) Cambium (d) Pith
6. Term meristem has been derived from:
(a) Meristos (b) Mitos
(c) Meritos (d) Merios
7. The meristem situated at the bases of internodes is:
(a)Apical Meristem (b) Intercalary Meristem
(c) Dermatogen (d) Lateral Meristem
8. Histogenic layer theory put forward by.
(a) Dermen (b) Foster
(c) Schmidt (d) Hanstein
9. Corpus is the:
(a) Central Zone (b) Peripheral zone
(c) Both (d) None
10. Protoderm cells are differentiated into:
(a) Vascular system (b) Epidermal systems
(c) Cambium (d) Cortex
11. Tunica-Corpus theory put forward by Schmidt in the year………….:
(a) 1924 (b) 1909

(c) 1960 (d) 1962

12. Histogen theory put forward by Hanstein in the year………….:
(a)1907 (b) 1868
(c) 1960 (d) 1990
13. The cells of tunica divides:
(a) Anticlinally (b) Periclinally
(c) Diagonally (d) All Planess
14. The cells of corpus divides in.
(a) All planes (b) Anticlinally
(c) Periclinally (d) Diagonally
15. Plerome gives rise to:
(a) Dermal system (b) Lateral Branch
(c) Vascular system (d) Leaf Primordia

(1) The term meristem originated from _______________.
(2) _________________proposed the Tunica-Corpus Theory.
(3) Histogen theory was put forward by __________.
(4) ________________ cells are actively dividing cells.
(5) Plerome gives rise to ________________ system.
(6) Dermatogen gives rise to ________________ system..
(7) Periblem gives rise to ________________ system..
(8) Histogenic layer theory put forward by__________________.
(9) The ___________________activity of cells provides growth and healing in plants.
(10) The _____________terminally positioned minute but complex structure that covered by
newly developing lateral organs like leaves or bracts.
(1) The term meristem originated from Greek term meristos.
(2) Peliblem gives rise to epidermal system.
(3) Tunica-Corpus theory was given by Schmidt.
(4) Histogen theory was discarded due to lack of cytological proof.

(5) According to Tunica-Corpus concept, the dividing cells in the apical meristem are arranged
in two zones.
(6) The meristem situated at the bases of internodes is Apical Meristem.
(7) The leaf primordia originate in the peripheral part of the apical meristem.
(8) Histogenic layer theory put forward by Dermen in 1947.
(9) The shoot apex consists of a set of meristematic cells in gymnosperms and angiosperms.
(10) Dermatogens consist the cells of the innermost layer and gives rise vascular system.
(1) Define meristems.
(2) Who proposed Tunica-Corpus theory?
(3) Who proposed Histogen theory?
(4) Define corpus.
(5) Define histogenic layers.
(6) What is the significance of periblem in plants?
(7) What is the anticlinal division?
(8) Describe anticlinal and periclinal division?
(9) Describe histogenic layers.
(10) Define plerome.
(11) Define Spurr’s media.
8.6.1 Answer keys: 1-b, 2-b, 3-b, 4-d, 5-a, 6-a, 7-b, 8-a, 9-a, 10-b, 11-a, 12-b, 13-a, 14-a, 15-c.
8.6.2 Answer key: 1-Meristos, 2-Schmidt, 3-Hanstein, 4-Meristmetic 5-Vascular, 6- Dermal,
7-Cortical, 8-Foster, 9- meristematic, 10- shoot apical meristem (SAM).
8.6.3 Answer key: 1-True, 2-False, 3-true, 4-True, 5-True, 6-False, 7-True, 8-True, 9-True,
10-False.
8.7 REFERENCES
 Clowes, F.A.L. (1961). Apical Meristems (Oxford: Blackwell Scientific Publications)

 Cutter, E.G. (1965). Recent experimental studies of the shoot apex and shoot
morphogenesis. Bot. Rev. 31, 7-113.
 Gilford, E. M. Jr (1954). The shoot apex in Angiosperm.” Bot. Rev,20, 477–529.
 Wardlaw, C.W. (1957). On the organization and reactivity of the shoot apex in vascular
plants. Am. J. Bot. 44, 176-185.
 Abraham Fahn.1990. Plant Anatomy. Oxford : Pergamon : Butterworth-Heinemann.

 Crang, Richard, Lyons-SobaskI, Sheila, Wise, Rober T. 2018. Plant Anatomy.A Concept-
Based Approach to the Structure of Seed Plants. Springer International Publishing,
 Katherine Esau. 1977. Anatomy of Seed Plants, 2nd Edition. A John Wiley & Sons.
 Ray F. Evert. 2006. Esau’s Plant Anatomy Meristems, Cells, and Tissues of the Plant
Body: Their Structure, Function, and Development (Third Edition). A John Wiley &
Sons, Inc., Publication.

(1) Define meristem.
(2) Write a note on Tunica Corpus theory.
(3) Describe SAM.
(4) What is Histogenic layer theory?
(5) Write a note on types of meristem.
8.9.2 Long answer question:
(1) Give a detailed account of SAM.
(2) Describe different theories put forward on sam.
(3) Write detailed note on tunica corpus theory.
(4) Give a illustrated account on apical meristem.
(5) Describe maceration techniques.

UNIT-9- EXAMINATION IN SHOOT APICES IN A

MONOCOT IN T.S. AND L.S. TO UNDERSTAND THE
ORIGIN OF LEAF PRIMORDIA.
9.1-Objectives
9.2-Introduction
9.3.1-Study of permanent/temporary slides showing SAM
9.3.1.1- Carex
9.3.1.2- Smilax
9.3.1.3- Datura
9.3.2-Study of permanent/temporary slides showing RAM

9.3.2.1- Allium
9.3.2.2- Nicotiana
9.3.3-Study of permanent/temporary slides showing the origin of leaf primordial in L.S. of shoot
apex
9.3.3.1- Triticum
9.3.3.2- Coleus
9.3.3.3- Saccharum
9.4-Summary
9.5-Glossary
9.7-References
9.1 OBJECTIVES
1. To examine the shoot apices in plant species
2. To study the root apices in plant species
3. To observe the anatomy of leaf primordia
9.2 INTRODUCTION
Shoot apex is the growing terminal of the stem. As shoot apex majorly consists of meristem
hence can also be called apical meristem. It is positively phototropic and negatively geotropic
and has dome shape. Apical meristem has terminal position in shoot apex. The meristematic cells
divide anticlinially near the Shoot Apical Meristems (SAMs). Shoot apex is generally protected

by young leaves forming a crown like structure lacks the quiescent centre and has differentiated
cells. The leaf primordium is present in the terminal part of the shoot.
Root apex, also called as root apical meristem is a portion of small region at the tip of a root in
which all cells are meristematic having high ability to division. The repeated division of cells led
to development of primary root tissues. Parenchymatic cells comprise the root cap which protects
the tip of roots while passing through the soil. Internal meristematic cells called calyptrogens
adds the new cells in the place of old ones and small root hairs arise as simple extensions from
root apex, increases the surface area for absorption of water and minerals from the soil.
Leaf primordial arises from foliar buttress which transform into axillar phyllopodium. The
meristem cells extend laterally. A highly ordered spatial and temporal pattern led to the
development of leaf primordial from the flanks of the SAM. As leaf expands, stomata
differentiate and gas spaces are formed.
9.3.1- Study of permanent/temporary slides showing shoot apex meristem (SAM)
9.3.1.1- L.S. of shoot apex of Carex (Fig 9.1 & 9.2)
1. The superficial tissue made up of rectangular cells forms a layer of tunica which is
uniseriate.
2. The central core of meristem called corpus is covered by the tunica mantle of cells.
3. The tunica initials are made up of a group of similarly appeared small group of apical
cells and the corpus is made up of two distinct zones.
4. The cells in the region of rib meristem which is the central region of the corpus, are
transverse vacuolated and rectangular in shape arranged with their long axis.
5. Elongated cells are present in the longitudinal extent of rib meristem of the species of this
genus.
6. The cells in flank meristem are smaller and less vacuolated than the other neighboring
cells which are partly vacuolated the rib meristem.
7. The cells of apical dome are large with vacuoles.
8. Few cells are larger in size than the rest and also more vacuolated than others.

Fig.9.3: Longitudinal sections of growing point of Carex shoot.
Fig.9.4: Longitudinal section of shoot apex of Carex hordeistichos.

9.3.1.2- L.S. of shoot apex of Smilax (Fig 9.3 & 9.4)
1. The species of this genus contain biseriate tunica present overlying a homogenous
corpus.
2. The cells of the shoot apex are highly stratified.
3. The primary meristem layer made up of meristematic cells is thick and radiates outward
below the apical meristem.
4. The meristematic cells are made up of small and nearly isodiametric cells.
5. The species of this genus are said to have true tunica and leaf initiation in subsurface
layers.

Fig.9.5: Longitudinal section of shoot tip of Smilax pumila.
Fig.9.6: Longitudinal section of shoot apical meristem of Smilax laurifolia.

9.3.1.3- L.S. of shoot apex of Datura (Fig 9.5)
1. The layer of tunica is made up of one or more layers of cells.
2. Three layers of apical meristem are present in which two layers are involved to form
tunica.
3. The central tissue of the shoot apex is made by third layer which constitutes to the initial
cells of the corpus.
4. The cells in periphery are made up of dividing meristematic cells.
5. The meristematic cells line up the marginal surface of leaf primordium.
6. Large vacuoles are present in the central core region of leaf primordium.

7. The polyploid and diploid cells are used to form the tissue of shoot meristem.
Fig.9.7: Longitudinal section of central core of primordial of Datura.

9.3.2- Study of permanent/temporary slides showing root apex meristem (RAM)
9.3.2.1- L.S. of root apex of Allium (Fig 9.6 & 9.7)
1. The cells of RAM are divided into different layers of epidermis, exodermis, central
cortex and endodermis.
2. The peripheral layer of root apex is the epidermis.
3. The cells made to form one celled thick layer called epidermis and they are closely
packed and elongated cells.
4. The cortex is divided into three parts which is comprised of exodermis, central cortex and
endodermis.
5. The outermost layer of cortex is called exodermis, whereas the innermost layer is called
endodermis whereas the space in between the exodermis and endodermis is occupied by
central cortex cells.
6. Casparian bands are present in the anticlinal cells of hypodermis to form exodermis
which is either dimorphic or uniform.
7. A dimorphic exodermis is made up of long and short cells whereas a uniform exodermis
is formed by uniform-shaped elongated cells.
8. Suberin lamella deposition in long cells acts as plasmodesmata.

9. The region of central cortex is made up of parenchyma cells which are large and loosely
arranged with numerous intercellular spaces.
10. The innermost tissue of the root apex is called stele which consists of pericycle, xylem,
and phloem and associated parenchyma cells.
Fig.9.8: The structure of root tip of Allium (https://dissectionconnection.com.au /shop/

downloads/labelled-microslides/plant-slides/allium-root-tip-100x/).

Fig.9.9: Longitudinal section of root apex meristem of Allium

(http://mrsmorrittscience.weebly.com/9-reactions-of-life-wk1.html).
9.3.2.2- L.S. of root apex of Nicotiana (Fig 9.8)

1. The root apex is made up of small group of cells.
2. Different layers or tiers comes together to form the tissue of root apex at the periphery.
3. Four types of initials naming root cap columella initials, the epidermis/peripheral root cap
initials, the cortex/endodermis initials and the vascular cylinder initials are present.
4. The initials are made up of three layers or tiers.
5. The lower tier is formed by the root cap, columella and epidermal cells.
6. The middle tier is made up of ground tissue whereas the upper tier is formed by vascular
cylinder.

Fig.9.8: The longitudional section of Nicotiana root apex meristem.
9.3.3- Study of permanent/temporary slides showing the origin of leaf primordia in L.S. of
shoot apex
9.3.3.1- L.S. of leaf primordial of shoot apex of Triticum (Fig 9.9)
1. Shoot apex somewhat resembles to convex structure
2. It is slightly elongated with broad bases
3. It is divided into three regions- two outer discrete and virtually self-perpetuating thimble
shells of cells (the dermatogens and the hypodermal layer)
4. Uniseriate hypodermal layer is present at the tip of apices
5. Vertical strips of chlorenchyma with small vasuclar bundles are observed in fig. 9.9.
6. Distinguishable hypodermal layer with periclinical breaks
7. Outer Parenchyma sheath is observed
8. New leaf primordium emerged from the periclinal region of dermatogens and
hypodermal layer.
9. The entire shoot apex is covered by dermatogen.

10. The uniform isodiametric or only slightly elongated non-vacuolated cells are arranged in
no striking arrangement in the central core.
11. At the apical part of the hypodermis, several large cells are arranged.
12. Under the cells of apical part lie small cells with capricious nuclei.
13. There is little distinction in cells at the tip of the apex.
Fig.9.9: L.S. of the Triticum leaf primordial.
9.3.3.2- L.S. of leaf primordial of shoot apex of Coleus (Fig 9.10 & 9.11)
1. Hairs like structures or projections are called Trichomes, covering the root surface.
2. The centre part of shoot apex is made up of small, closely packed cells known as Shoot
Apical Meristem (SAM).
3. Two leaf primordial are present on either side of SAM which later develops into leaves.
4. The centre of leaf primordial is made up of small cells of procambium which later
develops into vascular tissue.
5. The SAM is outlined by the protoderm cells.
6. The lignified cambium is present in cells.
7. Distinct xylem cells are present with lignified secondary wall.

8. The contracted protoplast is present is present in the xylem cells and hence it is called as
contracted xylem.
9. The pattern of differentiation of contracted xylem is not quite visible but distinction is bit
distinguished in distal region to the walled xylem.
10. Vascular cambium is evident due to the distinguished xylem parenchyma.
Fig.9.10:
The longitudinal section of shoot apex of Coelus (https://bio.libretexts.org/Bookshelves/Botany/
A_Photographic_Atlas_for_Botany_(Morrow)/12%3A_Stems/12.01%3A_Primary_Growth).

Fig.9.11: The closer view of Coleus shoot tip longitudinal section

(https://bio.libretexts.org/Bookshelves/Botany/A_Photographic_Atlas_for_Botany_(Morrow)/
12%3A_Stems/12.01%3A_Primary_Growth).
9.3.3.3- L.S. of leaf primordial of shoot apex of Saccharum (Fig 9.12)

1. The epidermal layer is present in the outermost surface called dermatogen.
2. The epidermis is interrupted by the stomata followed by hypodermis and then to sub-
hypodermis.
3. Next to the epidermal layer, ground tissue is present.
4. They hypodermal layer is consisting of the sclerenchyma or sclerified parenchyma cells.
5. The vascular bundles are scattered and embedded in to the ground tissue.
6. They lack endodermal layer and pericycle.
7. The smaller bundles are present in the periphery but the larger bundles are present in the
centre.
8. The bundles are collateral and closed, i.e., they lack cambium.
9. The vascular bundles are surrounded by the sclerenchymatous sheath.
10. The protoxylem and metaxylem are placed in the form of Y shape.

11. Phloem is made up of sieve tubes and companion cells.

12. The phloem lacks phloem parenchyma.
Fig.9.12: L.S. of Saccharum leaf primordial.
9.4- SUMMARY
Shoot apex consists of meristematic cells. The parenchymal cells are involved in forming the
outermost layer of shoot apex which is called epidermis. Cuticle covers the epidermal surface.
The L.S. of shoot apex is distinguished into the regions of tunica, corpus, flank meristem and rib-
meristem. The meristematic cells divide anticlinially whereas the cells of tunica region divide
periclinally. Trichomes or hairs are generally absent. Epidermis is followed by hypodermis
which is made up of sclerenchymatous cells. The vascular bundles contain xylem and phloem
but lack cambium.
Root apex has abundant meristematic cells which divide rapidly. The cells at the tip are protected
by root cap meristem cells which protects the growing tip. Similar to shoot apex, the outermost

layer is called epidermis which is followed by exodermis, central cortex and endodermis. These
different cells are arranged in various ties to form the tissue.
The leaf primordial on the shoot apex has a covering of epidermis made up of meristematic cells.
The epidermis is followed by the cells of ground tissue. The vascular bundles are present in
abundance and the structure supports the development of stomata structure within them.
9.5- GLOSSARY
Anticlinial- It means occurring at right angles to the surface or circumference of a plant organ
an anticlinal pattern of cell walls.
Biseriate- It is a botanical term meaning arranged in two rows.
Capricious- It refers to something which is unpredictable.
Flank- It means be on each or one side of.
Geotropic- It is the directional growth of an organism in response to gravity. Roots display
positive geotropism when they grow downwards, while shoots display negative geotropism when
they grow upwards. It is also called gravitropism.
Periphery- It refers the outer limits or edge of something.
Perennial- It means living for several years.
Periclinial- It means parallel to the surface of the meristem.
Perpetuate- To be continuing indefinitely.
Phototropic- Phototropism is the growth of an organism in response to a light stimulus.
Thimble- It is a small object with a closed end used for the protection.
Uniseriate- It refers the arrangement (of something) in single row.
9.6.- SELF ASSESSMENT QUESTIONS

1. The vascular bundles of Saccharum lack __________.
2. The meristematic cells of shoot meristem divide through ________division.
3. Suberin lamella deposition in long cells of root apex of allium acts as _.
4. The tunica layer of smilax is _________.
5. The tunica region of carex is ________.
6. The entire shoot apex of triticum is covered by ______.
7. The central region of shoot meristem of datura is called _______.
8. The peripheral region of the shoot meristem tissue of Datura is called ________.
9. The ________ is formed by inner protoxylem vessel and parenchyma break down in Zea.

10. Vascular cambium of coeus is evident due to the distinguished _______.
9.6.1- ANSWER TO THE SELF ASSESSMENT QUESTIONS

1. cambium
2. anticlinial
3. plasmodesmata
4. Biseriate
5. Uniseriate
6. dermatogens
7. corpus
8. tunica
9. xylem parenchyma
10. Sclerenchyma, sclerified parenchyma cells
9.7- REFERENCES
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27. Martin, B. F., & Tucker, S. C. (1985). Developmental studies in Smilax (Liliaceae). I.
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31. Satina, S., Blakeslee, A. F., & Avery, A. G. (1940). Demonstration of the three germ
layers in the shoot apex of Datura by means of induced polyploidy in periclinal
chimeras. American Journal of Botany, 27(10), 895-905.
32. Sharman, B. C. (1983). Developmental anatomy of the inflorescence of bread wheat
(Triticum aestivum L.) during normal initiation and when affected by 2, 4-D. Annals of
Botany, 52(5), 621-639.
33. Soukup, A., Seago Jr, J. L., & Votrubová, O. (2005). Developmental anatomy of the root
cortex of the basal monocotyledon, Acorus calamus (Acorales, Acoraceae). Annals of
Botany, 96(3), 379-385.
34. Stant, M. Y. (1952). The shoot apex of some monocotyledons: I. Structure and
development. Annals of Botany, 16(1), 115-129.
35. Tanaka, N. (2001). Taxonomic revision of the family Cannaceae in the New World and
Asia. Makinoa, new series, 1, 1-74.
36. Thomas, B., Murphy, D. J., & Murray, B. G. (2016). Encyclopedia of applied plant
sciences. Academic Press.
37. [USDA, ARS] US Department of Agriculture, Agricultural Research Service. (2011).
National Genetic Resources Program, Germplasm Resources Information Network
(GRIN) Online Database.
38. Waduwara, I. (2007). Onion root anatomy and the uptake of sulphate and phosphate
ions. University of Waterloo.

9.8- SUGGESTED READINGS

1. Lyndon, R. F. (1998). The shoot apical meristem: its growth and development.
Cambridge University Press.
2. Francis, D. (1999). The Shoot Apical Meristem. Its Growth and Development.
3. Dickison, W. C. (2000). Integrative plant anatomy. Academic press.
4. Maiti, R. (2012). Crop plant anatomy. Cabi.

1. What are the characteristics of root apices of Allium?
2. Describe the features of L.S. of shoot apices of Triticum.
3. Explain in detail about the L.S. of shoot apical meristem of Datura with well labelled
diagram.
4. Describe the leaf primordial of Saccharum.
9.9.1- Short Answer Type Questions
1. Draw the longitudinal section of primordial of Datura.
2. Give a brief detail of L.S. of Coleus leaf primordial.
3. What are the characteristics of shoot apex of Carex.
4. Describe the characteristics of L.S. of Smilax shoot apex.
9.9.2- Long Answer Type Questions
1. Provide a detail insight of anatomy of Triticum shoot apex.
2. Explain the anatomy of Allium.
3. Differentiate between the anatomical structure of Allium and Nicotiana with well-labeled
diagram.

UNIT-10 STUDY OF ALTERNATE AND DISTICHOUS,

ALTERNATE AND SUPERPOSED, OPPOSITE AND
SUPERPOSED OPPOSITE AND DECUSSATE LEAF
ARRANGEMENTS AND EXAMINATION OF ROSETTE
PLANTS AND INDUCTION OF BOLTINGS
10.1- Objectives
10.2- Introduction
10.3- Study of alternate and distichous, alternate and superposed, opposite and superposed
opposite and decussate leaf arrangements
10.4- Examination of rosette plants and induction of bolting
10.5- Summary
10.6- Glossary
10.7- Self Assessment Question
10.8- References
10.9- Suggested Readings
10.10- Terminal Questions

10.1 OBJECTIVES
 Understand the concept of phyllotaxy.
 Understand the bolting phenomenon in plants.
 Understand the alternate and distichous, alternate and superposed, opposite and
superposed, opposite and decussate leaf arrangements.
 Understand the rosette plants.
 Understand the induction of bolting.
10.2 INTRODUCTION
Phyllotaxy is the mode of arrangement of leaves on the stem. It is surprising how regular and
strictly mathematical this is. As a stem grows at its apex, new leaf buds form along the stem by a
highly controlled developmental process. Leaf arrangement along the plant stem depends on the
species and is an important identifying characteristic. The function of the arrangement of leaves
(phyllotaxy) is to increase a plant’s ability to carry on photosynthesis and other physiological
activity like transpiration etc, it is done by positioning the leaves in such manner to maximize the
surface area available to capture radiation. Leaves may be either caulescent (with obvious stems)
or acaulescent (without obvious stems). Leaf is a flattened lateral appendage of stem specialized
for food production. On the basis of development and origin leaves may be categorized as:
i. Seed leaves or cotyledons present on the seed.
ii. Foliage leaves: Flat, green, lateral appendages developing on stem or branch.
iii. Scaly leaves: Scale like, small, stalkless, non- green leaf. These are also called cataphylls.
iv. Bract leaves or hypsophylls: These are present at the base of flower of inflorescence.
v. Prophylls: These are bracteoles.
vi. Ligules: These are tongue like small parts present at the upper end of leaf sheath.
vii. Floral leaves: Calyx, corolla, stamens and carpels.
viii. Sporophylls: Spore bearing leaves.
Leaves are arranged on the stem in a definite manner of a particular species which are controlled
genetically. The arrangement is always in regular manner by obeying order of genes and leaves
are never placed on the stem in a haphazard manner. The foliage leaves are usually produced on

the axis with long or short internodes between them. This arrangement is called cauline. In some
cases, however, the leaves arise in a cluster from the very short stem just on the top of the root
e.g. carrot, radish etc called rosette. This arrangement is called radical although the leaves do not
actually arise from the root but from a very much condensed stem as in the case of radish, turnip
etc. In cauline leaves there may be one, two, three or more leaves at each node. On this manner
phyllotaxy is divided in two categories: alternate or acyclic and cyclic. When there is only one
leaf attached on the node the arrangement is spiral or alternate or acyclic. When there are two or
more leaves arranged at each node of the stem then phyllotaxy is called cyclic.
10.3 STUDY OF ALTERNATE AND DISTICHOUS, ALTERNATE

AND SUPERPOSED, OPPOSITE AND SUPERPOSED OPPOSITE
AND DECUSSATE LEAF ARRANGEMENTS
Leaves arrange on the stem in two manners thus forms two types of phyllotaxy: (1) Cyclic
Phyllotaxy and (2) Spiral or Alternate or Acyclic Phyllotaxy.
A. Cyclic Phyllotaxy:
In the cyclic type of phyllotaxy the leaves at each node form a whorl with the leaves placed on a
circle in which the angles between adjacent leaves are the same. Cyclic phyllotaxy includes both
opposite and whorled type phyllotaxy.
(I) Opposite Phyllotaxy: According to Snow and Snow in opposite phyllotaxy the two leaves at
each node are arranged as opposite to one another. If the successive pairs of leaves be placed at
right angles to one another, the arrangement is termed opposite decussate. In this case, when
looked from the top, all the leaves will be found to be arranged along four vertical rows. This is
found in Calotropis, Gardenia, Ixora, Ocimum, Mentha etc. In some other plants it is found that
the successive pairs are placed exactly on top of one another so that all the leaves lie in one plane
and when viewed from above all the leaves are found to lie in two vertical rows. This type of
phyllotaxy is known as opposite superposed . It should be noted that while the superposed type is
found in many plants like Psidium gujava (guava), Combretum etc., in many instances the
phyllotaxy is initially decussate which later becomes superposed by the flattening out of the
twig.
(II) Verticillate Phyllotaxy or Whorled Phyllotaxy:
Oleander plant (Nerium indicum) shows three leaves forming a whorl at each node while
Saptparni (Alstonia scholaris) shows five or more leaves. These are instances of the verticillate

type. Sometimes verticillate phyllotaxy is designated as whorled phyllotaxy. Thus, if there be

two leaves in a whorl, the two will be placed opposite (i.e., at an angular distance of two right
angles) one another. If there be three leaves, the angle between leaves in the same whorl is 120°
(i.e., one third of a circle), if four, it is 90° and so on.
Fig.1 (A-D): Different types of phyllotaxies

B. Spiral or Alternate or Acyclic Phyllotaxy:
This is the common type phyllotaxy found in plants and the mathematical regularity of the
arrangement is astonishing. One can easily verify that (1) the angular distance (angular
divergence) between any two consecutive leaves is constant; (2) when looked from the top, all
the leaves are found to lie in a fixed number of vertical rows or orthostichies; (3) the vertical
lines (hypothetical) are evenly dispersed on a circle, the angle between adjacent orthostichies
being constant; (4) although leaves look scattered, a close examination shows that leaves are
evenly dispersed on all sides of the stem. In spiral phyllotaxy one has to imagine a line touching
the bases of successive leaves. It will be found that this line forms a spiral (hence the name spiral
phyllotaxy) on the stem. This spiral is called the genetic spiral. This genetic spiral can easily be
projected on a flat surface to form a fiat spiral and the position of the leaves may be marked on
this spiral. The angle subtended at the centre by two consecutive leaves is the angular
divergence. In practice, the angular divergence may be denoted by finding out a leaf which is
exactly above a particular leaf, then by counting the number of complete circles covered by the
genetic spiral and the number of leaves beginning from the first to the one just before the last.
The latter number of leaves-also gives the number of orthostichies. Thus, sometimes, only the
above fraction is given without calculating the actual divergence in degrees.
(1) Distichous or ½ Phyllotaxy:

In the family Poaceae (e.g., Cynodon, Oryza) the leaf on the second node is just opposite of the
leaf on the first node, the third leaf is above the first leaf, the fourth above the second and so on.
Thus, the angular divergence is clearly 180° (straight angle) and all the leaves fall in two
opposite orthostichies (hence, the name distichous). The genetic spiral covers only one circle and
there are two leaves on the way in coming to the leaf exactly above the first leaf. Hence, the
angular divergence = 1 (Circle)/2 (orthostichies) of 360º = 180º. As only the fraction is usually
given, the phyllotaxy is also called ½ phyllotaxy.
(2) Tristichous or 1/3 Phyllotaxy:

In Cyperus rotundus (Sedge) the leaves are three-ranked (three orthostichies); the fourth leaf is
above the first leaf, fifth above second, sixth above third and so on. Three leaves lie in one circle
of the genetic spiral. Hence, angular divergence = 1/3 of 360° = 120° or 1/3 phyllotaxy.
(3) Pentastichous or 2/5 Phyllotaxy:

In some plants like the Hibiscus and Ficus bengalensis, the sixth leaf is found above the first
leaf, seventh leaf above the second one and so on. The genetic spiral completes two circles in
passing these five leaves or five orthostichies . Hence, angular divergence = 2/5 of 360° = 144°
or 2/5 phyllotaxy.
(4) Octastichous or 3/8 Phyllotaxy:

In this case the ninth leaf is found above the first leaf and the genetic spiral completes three
circles in this distance as in the case of the Carica papaya of family caricaceae. Thus, there are
eight orthostichies. The angular divergence is 3/8 of 360°= 135º.
It is rather interesting to note that in the common types of phyllotaxy the fractions of angular
divergence lie in a series in which the numerator and the denominator in each case are obtained
by adding up the numerators and denominators of the two preceding phyllotaxies. Thus, ½,
This series is called the fibonacci series or the Schimper-Brown series of

divergence. When the divergence becomes a big fraction it is not possible to count the
orthostichies.
In plants showing these phyllotaxies, usually the internodes are very short so that the leaves are
much crowded together. On date and other palms with persistent leaf bases showing such
complex phyllotaxy one very close spiral of leaf bases is observed. This type of phyllotaxy is
called parastichous . The sporophylls on a pine cone are also arranged in the same way and the
parastichous arrangement can be seen clearly when the cone is viewed from above.
Besides the fibonacci series, which is by far the commonest, there are possibly some other series
of phyllotaxy. Two such rare series 1/4, 1/5, 2/8, 3/14…. and 1/2, 2/8, 2/5, 5/8, 8/18…The laws
of phyllotaxy may sometimes be disturbed by some twisting of the stem or by incomplete
development as towards the apex of a twig.
10.4 EXAMINATION OF ROSETTE PLANTS AND INDUCTION

OF BOLTING
A rosette is manner of leaf arrangement of plants in which leaves or of structures resembling
leaves arrange in a circular manner. In angiospermic plants, rosettes usually sit near the soil.
Rosette structure is an example of a modified stem in which the internodes are highly
compressed and gaps between the leaves do not expand, so that all the leaves remain clustered
tightly together and at a similar height. If a plant grows in a rosette form, the leaves will radiate
from the center stalk either right at ground level or close to the ground. The term rosette is used
because the pattern resembles the habit of a rose’s flower. Many types of plant grow in a rosette
pattern.
Generally rosettes are found in perennial plants whose upper foliage dies back with the
remaining vegetation protecting the plant. Many perennials begin life as a rosette but progress to
become more shrub like. Perennials and biennials benefit from the early rosette formation
because it exposes as many leaves as possible to the sun, while maintaining a low profile to
avoid being eaten by browsing animals. Most succulents that form rosettes maintain that form
their entire lives. The rosette formation allows for maximum exposure to the sun while allowing
the plants to capture and direct moisture toward the roots. Most succulents come from arid areas
where thick leaves allow them to retain water.
Another form occurs when internodes along a stem are shortened, bringing the leaves closer
together, as in Lactuca sativa (lettuce), Taraxacum officinale (dandelion) and some succulents.

When plants such as lettuce grow too quickly, the stem lengthens instead, a condition known as
bolting. In some cases, the rosette persists at the base of the plant (such as the dandelion), and
there is a taproot system.
Bolting is the elongation of internodes or production of a flowering stem. These flowering stems
are usually vigorous extensions of existing leaf-bearing stems, and in order to produce them, a
plant diverts resources away from producing the edible parts such as leaves or roots, resulting in
flavor and texture changes, withering, and in general, a poor quality harvest. Plants that have
produced flowering stems in this way are said to have bolted. Crops inclined to bolt include
Lactuca sativa (lettuce), Beta vulgaris (beetroot), Brassicas, Alium (onion) etc.
According to Rood et. al.,(1989) bolting is induced by plant hormones of the gibberellin family,
can occur as a result of several factors, including changes in Photoperiod (day length), the
prevalence of high temperatures at particular stages in a plant's growth cycle and the existence of
stresses such as insufficient water or minerals. These factors may interact in a complex way.
According to Zeevaart (1971) photoperiod may affect the propensity to bolt thus some plants are
"long day plants", some are "short day plants" and some are "day neutral", for example when a
long day plant, such as spinach, experiences increasingly long days that reach a particular length,
it will be inclined to bolt. Low or high temperatures can affect the propensity of some plants to
bolt if they are experienced for sufficient periods at particular points in the life cycle of the plant;
once these conditions have been met, plants that require such a trigger will subsequently bolt
regardless of subsequent temperatures. Plants under stress may respond by bolting so that they
can produce seeds before they die.
The essential cause of plant blotting is the premature production of a flowering stems on a plant
before it can be harvested. Bolting is the plant's natural attempt to produce seeds so it can
reproduce. It occurs when the weather heats up. The extensions are its leaf-bearing parts. Bolting
is also known as going to seed. The term bolting is typically used with regard to vegetable
gardening. Most of plants bolt due to hot weather. When the ground temperature goes above a
certain temperature, this flips a switch in the plant to produce flowers and seeds very rapidly and
to abandon leaf growth almost completely.
Gibberellins stimulate stem growth in dwarf and rosette plants. Applied gibberellin promotes
elongation of internodes in a wide range of plant species. However, the most dramatic
stimulations are seen in dwarf and rosette species, as well as members of the grasses. Exogenous

gibberellin causes such extreme stem elongation in dwarf plants that they resemble the tallest
varieties of the same species. Accompanying this effect are a decrease in stem thickness, a
decrease in leaf size, and a pale green color of the leaves. Some plants assume a rosette form in
short days and undergo shoot elongation and flowering only in long days. Gibberellin application
results in bolting in plants kept in short days, and normal bolting is regulated by endogenous
gibberellin especially rosette species. Gibberellin is thus a component of the flowering stimulus
in some plants.
10.5 SUMMARY
1. Phyllotaxy is the mode of arrangement of leaves on the stem. It is surprising how regular and
strictly mathematical this is.
2. Leaf arrangement along the plant stem depends on the species and is an important identifying
characteristic.
3. The function of the arrangement of leaves (phyllotaxy) is to increase a plant’s ability to carry
on photosynthesis and other physiological activity like transpiration, etc.
4. Leaves may be either caulescent (with obvious stems) or acaulescent (without obvious
stems). Leaf is flattened lateral appendage of stem specialized.
5. In some cases, however, the leaves arise in a cluster from the very short stem just on the top
of the root e.g. carrot, radish etc., called rosette.
6. Leaves arrange on the stem, two manners thus forms two types of phyllotaxies: (1) Cyclic
Phyllotaxy and (2) Spiral or Alternate or Acyclic Phyllotaxy.
7. In the cyclic type of phyllotaxy the leaves at each node form a whorl with the leaves placed
on a circle in which the angles between adjacent leaves are the same.
8. A rosette is a manner of leaf arrangement of plants in which leaves or of structures
resembling leaves arrange in a circular manner.
9. Rosette structure is an example of a modified stem in which the internodes are highly
compressed and gaps between the leaves do not expand, so that all the leaves remain
clustered tightly together and at a similar height.
10. Generally rosettes are found in perennial plants whose upper foliage dies back with the
remaining vegetation protecting the plant.
11. When plants such as lettuce grow too quickly, the stem lengthens instead, a condition known
as bolting.

12. Bolting is the elongation of internodes or production of a flowering stem

13. According to Sood et. al., bolting is induced by plant hormones of the gibberellin family and
can occur due to several factors, including changes in photoperiod (day length), the
prevalence of high temperatures at particular stages in a plant's growth cycle, and the
existence of stresses such as insufficient water or minerals.
10.6 GLOSSARY
Phyllotaxy: Arramgement or pattern of leaf attachment on stem.

Bolting: Elongation of internode or floral axis.
Cataphyll: A simplified leaf form, as a bud scale or a scale on a cotyledon or rhizome.
Cauline: Leaves growing on a stem especially on the upper part of a stem.
Cotyledons: Primary or rudimentary leaf of the embryo of seed plants.
Gibberellins: Gibberellins are plant hormones that regulate various developmental processes,
including stem elongation.
Hypsophylls: A floral leaf beneath the sporophylls.
Ligule: A thin, membranous outgrowth from the base of the blade of most grasses
Perennial: Perennial plants are those that continue to grow year after year.
Rosette: A leaf rosette is a plant growth habit in which a plant grows a cluster of leaves in a
circular pattern.

1. The bolting in plants caused by
(a) Gibberellins (b) Auxins
(c) Cytokinins (d) Ethylene
2. Premature stem elongation of plants is called

(a) Vernalisation (b) Bolting

(c) Elongation (d) Flowering
3. Arrangement of leaves on stem is called

(a) Aestivation (b) Phyllotaxy
(c) Inflorescence (d) Phototaxis
4. The leaves arise in a cluster from the very short stem just on the top of the root
(a) Cyclic (b) Opposite
(c) Rosette (d) None of these
5. Photoperiod is associated with which phenomenon in plants

(a) Phototaxis (b) Bolting
(c) Phyllotaxy (d) All
6. Primary or rudimentary leaf of the embryo of seed plants

(a) Cataphylls (b) Cotyledons
(c) Sporophylls (d) None of the above
7. Spore bearing leaves

(a) Cataphylls (b) Cotyledons
(c) Sporophylls (d) None of the above
8. Taraxacum officinale leaves are example of

(a) Opposite phyllotaxy (b) Rosette
(c) Spiral phyllotaxy (d) Cyclic phyllotaxy
9. The other name for fibonacci series ( ) is
(a) Schimper-Brown series (b) Goldman series

(c) Lehman series (d) Fabnori series

10. Bolting can be induced by hormone

(a) Auxin (b) Cytokinin
(c) Ethylene (d) Gibberelins

(1) Premature stem elongation in plants is called _______________.
(2) Photoperiod is associated with ________________ phenomenon in plants.
(3) Bolting can be induced by hormone __________.
(4) This series in phyllotaxy is called the ‘Fibonacci Series’ or
the________________.
(5) Spore bearing leaves are called ________________.
(6) 3/8 phyllotaxy is also called_______________.
(7) Pentastichous is _____________ phyllotaxy.
(8) ______________structure is an example of a modified stem in which the internodes are
highly compressed.
(9) Arrangement of leaves on stem is called _____________.
(10) Oleander plant (Nerium indicum) shows three leaves forming a whorl, such type of
phyllotaxy is called____________________.
10.7. 3 True or False:

(1) Bolting can be induced by auxin hormone.
(2) Pentastichous is 3/8 phyllotaxy.
(3) Premature stem elongation of stems of plants is called rosette.
(4) Premature stem elongation of stems of plants is called bolting.
(5) Photoperiod is associated with bolting phenomenon in plants.
(6) Taraxacum officinale (Dandelion) leaves are example of verticellate phyllotaxy.
(7) Oleander plant (Nerium indicum) shows three leaves forming a whorl; such type of
phyllotaxy is called verticellate phyllotaxy.
(8) 3/8 phyllotaxy is also called octastichous.

(9) A rosette is manner of leaf arrangement of plants in which leaves or of structures resembling
leaves arrange in a circular manner.
(10) Bolting is induced by plant hormones of the gibberellin family.

(1) Define bolting.
(2) Which plant hormone is called bolting hormone?
(3) What is phyllotaxy?
(4) Define Schimper- Brown series in phyllotaxy.
(5) Define rosette habit of plants.
(6) What type of phyllotaxy is found in Oleander (Nerium) plant?
(7) Define orthostichies.
(8) What do you mean by octastichous condition?
(9) Define sporophylls.
(10) Define cataphylls.
10.7. 1 Answer key: 1-(a), 2-(b), 3-(b), 4-(c), 5-(b), 6-(b), 7-(c), 8-(b), 9-(a), 10-(d).
10.7.2 Answer key: 1-Bolting, 2-Bolting, 3-Gibberellin, 4-Schimper-Brown series 5- Sporophylls,
6- Octastichous, 7- 2/5, 8-Rosette, 9-Phyllotaxy, 10- Verticellate.
10-True.
10.8 REFERENCES
 Rood B. S, Pearce D, Williams P.H and Pharis R. P. (1989). A gibberellin-deficient Brassica

mutant-rosette, Plant Physiology. 89:482-487.
 Snow, M. and Snow, R. (1934). The interpretation of Phyllotaxis. Biological Reviews. 9 (1):
132-137.
 Zeevaart, J. A. D. (1971). Effects of Photoperiod on Growth Rate and Endogenous
Gibberellins in the Long-Day Rosette Plant Spinach. Plant Physiology. 47 (6): 821–827.


 Roger V. Jean. 1994. Phyllotaxis : A Systematic Study in Plant Morphogenesis. Cambridge
[England]; Cambridge University Press. New York.
 Lincoln Taiz and Eduardo Zeiger 2002. Plant Physiology, 3rd ed. Sinauer Associates
Sunderland.

(1) Write short note on phyllotaxy.
(2) Describe rosette habit with examples.
(3) What do you understand by bolting?
(4) Describe Brown-Schimper series.
(5) Differentiate between octastichous and pentastichous condition.
(6) Differentiate between opposite and alternate phyllotaxy.
(7) Write short note on bolting.
(8) What do you understand by pentastichous condition?
(9) Write a note on bolting.
(10) Define cataphylls.

(1) Describe phyllotaxy in detail.
(2) Differentiate between cyclic phyllotaxy and spiral phyllotaxy.
(3) Write a detailed note on ostastihies.
(4) Write a note about bolting and its cause.
(5) Describe cyclic phyllotaxy and its types.

UNIT-11 MICROSCOPICAL EXAMINATION OF

VERTICAL SECTION OF LEAVES
11.1- Objectives
11.2- Introduction
11.3- Microscopical examination of vertical section of leaves
11.3.1- Gymnosperms
11.3.1.1- Cycas
11.3.1.2- Pinus
11.3.2- Dicotyledons
11.3.2.1- Ficus
11.3.2.2- Nerium
11.3.2.3- Eucalyptus
11.3.2.4- Pyrus
11.3.3- Monocotyledons
11.3.3.1- Phoenix
11.3.3.2- Allium
11.3.3.3- Musa
11.3.3.4-Triticum
11.3.3.5-Oryza
11.3.3.6-Zea
11.3.3.7-Bambusa
11.4- Summary
11.5- Glossary
11.6- Self Assessment Question
11.7- References

11.1 OBJECTIVES
1. To examine the vertical section of leaves microscopically.

2. To know about anatomically features of gymnosperms, dicot and monocot leaves
11.2 INTRODUCTION
The leaves in collection are called foliage, are the organ of vascular plant. It provides the major
site for photosynthesis. Usually, the primary photosynthetic tissue, the palisade mesophyll is
present in the upper side of the blade or lamina of leaf. Leaves can be petiolated or sessile.
Vertical section of leaf contains stomata openings by which oxygen and carbon dioxide can be
exchanged between plant and the atmosphere. Usually, the lower surface of leaf contains highest
number of stomata in its epidermal layer. These openings in the epidermal layer contribute to the
process of photosynthesis by exchanging gases and to block the transpiration so that loss of
water can be minimized.
11.3.1-Gymnosperms
11.3.1.1-Cycas
Cycas belongs to the very ancient lineage of plants. They were maximum diverse around the
Jurassic and Cretaceous periods. They are evergreen perennials and contain 113 species. One of
the species C. circinalis is an endemic species in India and it is the first cycad species to be
described in western literature. The species of this genus do not belong to the palms, ferns, trees
or any other modern group of plants. Most of the species of Cycas became extinct during the
extinction of non-avian dinosaurs.
Habit and Habitat: Cycas is known to originate from the medieval time and hence correct
region of origin is difficult to determine. Most of the species are known to become extinct with
the extinction of non-avian dinosaurs. These plants are abundant in the equatorial regions of
eastern and southeastern Asia including the Philippines with 10 species (out of which 9 are
endemic), eastern Africa (including Madagascar), northern Australia, Polynesia and Micronesia.
Australia inhabits 26 species while the Indo-Chinese area inhabits 30 species. Also, India has 9
species. The northernmost species C. revoluta is found at 31°N in southern Japan. The
southernmost C. megacarpa is found at 26°S in southeast Queensland. Due to the occurrence of

large number of Cycas species in China, Australia and India, these countries are considered as
centres of Cycas diversity.
V.S. of Cycas leaf (Fig 11.1)
1. The epidermis is the outermost layer which consists of cuticularised thick-walled cells.
2. The upper epidermis is continuous but lower epidermis is interrupted by the presence of
sunken stomata.
3. The stomata are haplocheilic with two guard cells and 8-10 subsidiary cells arranged in
ring in the guard cells border.
4. The 1-2 layered (3-4 layered at the margins) thick hypodermis is present below the
epidermis which is made up of cholrenchyma and sclerenchyma.
5. The hypodermis is followed by the parenchymatous ground tissue with many mucilage
canals.
6. The mesophylls are divided into the palisade and spongy parenchyma.
7. The palisade cells are present below the hypodermal layer and below the palisade cells;
spongy parenchymatous cells are placed with intercellular spaces.
8. The vascular bundles are diploxylic and they consist of triangular centripetal exarch
metaxylem and centrifugal endarch primary xylem.
9. The 3-4 layers of tracheid cells are present on either side of the centripetal metaxylem, in-
between the upper palisade layer and lower spongy layer.
10. The phloem is present on abaxial side below the xylem.
11. The phloem constitutes from the sieve and parenchymatous cells.
Fig.11.1: V.S. of Cycas revoluta leaf

11.3.1.2-Pinus
The Pinus belongs to the Pinaceae family, are conifers which contains 126 species names of
pines, together with 35 unresolved species. They are also referred by lumber, derived from the
pine trees. It is the sole genus which belongs to Pinoideae subfamily. All the species are listed in
the Royal Botanic Gardens Kew and Missouri Botanical Garden.
Habit and Habitat:
Pines are native to the Northern Hemisphere and some parts of Southern Hemisphere. One of the
species named, Sumatran pine crosses the equator in Sumatra to 2oS. They are found in the far
north as 66on to as far south as 12 oN, in the regions of North America. Although, pines are found
in variety of environmental conditions which are ranged from semi-arid desert to rainforests,
from sea level up to 5200 meters. They are dispersed from cold to hot environments on Earth.
Some species are also introduced in temperate and subtropical regions of both hemispheres. They
are also used as ornamental plants in parks and gardens.
V.S. of Pinus leaf (Fig 11.2)
1. It features a thick-walled epidermis with a single layer, dense cuticle.
2. Depressed stomata run the length of the surface (amphistomatic).
3. There are two guard cells and two subsidiary cells in each stoma.
4. It has two openings: one on the outside, termed the vestibule, and one on the interior,
called the substomatal cavity.
5. Providing structural rigidity, a few layers of dense sclerenchymatous hypodermis are
found beneath the epidermis.
6. Palisade and spongy parenchyma are not distinguishable.
7. Thin-walled, parenchymatous, polygonal, compactly packed cells with chloroplasts and
starch grains make up one such section.
8. The inner surface has peg-like infoldings.
9. Single-layered endodermis with barrel-shaped cells and casparian strips surrounds it.
10. A multilayered pericycle with a T-shaped mass of sclerenchymatous cells amid two
vascular bundles lies beneath endodermis.
11. Somewhat on side, there is transfusion tissue.

12. There are three types of bundles: collateral, open, and endarch.
13. Xylem is divided into tracheids but it lacks vessel members.
14. The parenchyma cells are modified to form albuminous cells.
15. The phloem tubes do not have direct interaction with companion cells.
16. The vessels are enclosed in transfusion tissue.
17. The vessels are consisting of parenchyma cells and tracheids.
18. The mesophyll cells are present in abundant and differentiated into the separate palisade
and spongy mesophylls.
19. Casparian strips or suberin are present in endodermis layer at young stage of the leaf.
20. Suberins are arranged in radial fashion to create a barrier for water movement in the
apoplast.
Fig.11.10: V.S. of Pinus roxburgii leaf

(https://www.studyadda.com/notes/11th-class/biology/plant-kingdom/some-representative-
rymnosperms/9569).
11.3.2-Dicot
11.3.2.1-Ficus

Ficus are the woody trees, shrubs, vines, epiphytes and hemiepiphytes. They belong to the
Moraceae Family. It contains 850 species in total. They are widely known as Fig Trees or Figs.
The fruits of most these species are edible but they are of low economic importance to human
and eaten as bush food. Regarding wildlife, they are considered as the extremely important food
resources. They are also regarded as objects of worships and for many practical uses.
Habit and Habitat: Figs are abundant in Tropical Forest ecosystems. They are known to be
native from the regions of the southwest Asia and the Mediterranean region, i.e., from
Afghanistan to Portugal. They are known to carry great importance for wildlife by use by
frugivores such as fruit bats and primates which include capuchin monkeys, langurs, gibbons and
mangabeys. Also, birds like Asian barbets, pigeons, hornbills, fig-parrots and bulbuls are entirely
dependent on figs when they are in abundant to survive.
V.S. of Ficus leaf (Fig 11.3)
1. The epidermis is composed of oval cells with undulating walls.

2. The mesophyll cells can be bifacil and isobilateral.
3. 3-5 rows of ordinary parenchyma cells are arranged in spongy layers.
4. The aerenchyma cells are also present in spongy layers.
5. The mid-rib cross-section is in quadrilateral shape.
6. The crescent shaped vascular bundles are present.
7. The vascular bundles are divided into regions of phloem and xylem.
8. Several layers of sclerenchyma cells are arranged around the vascular bundles.

Fig.11.3: V.S. of Ficus elastica leaf (Shakir and Baji, 2016).

11.3.2.2- Nerium
Nerium contains the only single species, N. oleander. It is a small tree or shrub, which belongs to
the Apocynaceae family. It is a widely cultivated plant. Its origin is supposed to be the regions of
southwest Asia. This plant is also known for its poisonous properties but used as ornamentally
grown garden plant.
Habit and Habitat: Nerium is native to the regions of Mauritania, Morocco and Portugal
eastward through the Mediterranean region and the Sahara, to the Arabian Peninsula, Southern
Asia and Southern parts of China. It is widely dispersed plant around the subtropical and tropical
areas of the world. It is known to be planted around the stream beds in river valleys so that it can
tolerate long seasons of drought and deluge from winter rains.
V.S. of Nerium leaf (Fig 11.4)
1. The leaf is dorsiventral and consists of upper and lower epidermis.

2. A thick cuticle coats the outside of leaf surface.
3. Cuticle is followed by the multi-layered (three-layered) upper epidermal cells and
generally stomata are not found on this surface.
4. Two kinds of mesophyll cells are present which are called palisade and spongy cells.
5. The lower epidermis consists of a single row of barrel shaped cells, closely arranged and
protected by cuticle.

6. The stomata are found in the depressions formed due to incurve of the lower epidermis.
7. Stomata are protected with trichomes.
8. Vascular system is well-developed.
9. Palisade cells are situated below the epidermal cell layers.
10. Spongy tissue is followed by palisade tissue.
11. Spongy cells are arranged in loose fashion due to which many intercellular spaces are
present.
12. The rosette crystal within leaf cell is present in between the spongy tissue, called druses.
13. Stomata is placed in the pit and protected by trichomes.
14. Lower epidermal cells are lined by thin cuticle.
15. Above the lower epidermis, palisade parenchyma is present.
Fig.11.11: V.S. of Neriumleaf (https://brainly.in/question/43171596).
11.3.2.3-Eucalyptus

Eucalyptus is a genus of plant comprising over seven hundred species of flowering trees, shrubs
or mallees. It belongs to the myrtle family, Myrtaceae. They are commonly called as Eucalypts
with the other genera in the tribe Eucalypteae. This plant has much commercial importance. Its
bark can either be smooth or fibrous, hard or stingy. The leaves contain oil glands, sepals and
petals are fused together to form cup-like structure over the stamens, called operculum. The
fruits are known as “Gumnut”. They are known to be native to Australia but it is cosmopolitan in
distribution.
Habit and Habitat: Eucalyptus are found in varying sizes. They can be diverse from different
shrubs to tall trees. As a shrub, they are mature plant with 1 meter height and grow in an extreme
environment. They are mainly the plants of cold tolerance.
V.S. of Eucalyptus leaf (Fig 11.5)
1. The single-layered epidermis covers the upper and lower surface of the leaf.
2. Mesophyll cells are arranged into two categories naming, palisade parenchyma and
spongy parenchyma.
3. Palisade parenchyma cells are situated both below and above the upper and lower
epidermis, respectively.
4. The layer of spongy parenchyma is placed between the palisade parenchyma.
5. The spongy parenchyma is arranged in loose fashion inside the cells due to the presence
of many intercellular spaces in between.
6. The calcium oxalate crystal is placed in between the spongy parenchyma layer

Fig.11.12: V.S. of Eucalyptus leaf
11.3.3.4-Pyrus
Pyrus belongs to the Rosaceae family which bears the pomaceous fruits. Most of the fruits
belonging to this genus are edible. The tree is of medium-size and is known to be the native to
coastal as well as mildly temperate regions of Europe, North Africa and Asia. This plant is
known to be of great importance in manufacturing high-quality woodwind instruments and
furniture. They are known to be native of central and Eastern Europe and southwest Asia. They
are mostly grown in temperate regions of Europe, North America and Australia. Two species
called P. pyrifolia and P. bretschneideri are widely grown in East Asia.
Habit and Habitat: Pyrus is native to the coastal and temperate regions of Europe, North Africa
and Asia. It is distributed across the Himalayas, from Pakistan to Vietnam and southern province
of China to the northern region of India. It is also dispersed in Kashmir, Iran and Afganishtan.
They are tolerant trees which grow in sandy loamy soil with well drainage. They are adaptive
towards 750-1500 mm/yr or more precipitation zone and 10-35oC temperature.
V.S. of Pyrus leaf (Fig 11.6)
1. The single-layered epidermis covers the upper and lower surface of the leaf.

2. Mesophyll cells are divided into two groups called as Palisade Parenchyma and Spongy
Parenchyma.
3. The bundle sheath extensions are present beneath the epidermis
4. The vascular bundles are present in form of xylem and phloem with the fibers
5. The palisade parenchyma is placed beneath the upper epidermis in loose fashion
6. The spongy parenchyma is situated above the lower epidermis in a very loose manner
Fig.11.13: V.S. of Pyrus leaf

11.3.3-Monocot
11.3.3.1- Phoenix
It is the genus containing 14 species of palms. Genus is native to Canary Islands in the west,
across northern and central Africa, to the extreme southeast of Europe (Crete) and then covering
the southern Asia from Turkey east to southern China and Malaysia. They are found in swamps,
deserts and mangrove sea coasts. They have been originated in semiarid region but are usually
found in high groundwater levels, rivers or springs. Plants were more abundant and widespread
in the past than they are now,
Habit and Habitat: In Iran, Iraq, Arabia, and North Africa west of Morocco, this genus
comprises species that are important. They are also cultivated in America in southern California,
Arizona and southern Florida in the United States and in Sonora and Baja California in Mexico.

They are grown in variety of soils, in the degraded forest margins in grasslands, even grown
under the shade of dominating forest trees along fragile hill slopes and stream courses in warm
and humid conditions.
V.S. of Phoenix leaf (Fig 11.7)
1. The epidermis is bilayer and made up of compactly arranged tabular cells.
2. The cuticularized walls are present on them.
3. Stomata are scattered throughout the epidermal layer.
4. Just internal to both epidermal layers, a layer of parenchyma cells is present with scanty
chlorophyll.
5. A special hypodermis as a subepidermal layer is observed in microscopy.
6. Mesophyll is made up of more or less isodiametric cells with small intercellular spaces.
7. Absence of palisade and spongy cells differentiation is observed.
8. Sclerenchyma patches are present in more or less parallel series towards the both upper
and lower epidermis.
9. The bundles are collateral and closed and are arranged in parallel series.
10. Xylem is placed on the upper and phloem is situated on the lower side.
11. The large bundle contains the heavily thick-walled sclerenchyma patches on both the
sides.
12. The small bundles are surrounded by parenchyma sheath with no chlorophyll.

Fig.11.7: V.S. ofPhoenix leaf

11.3.3.2- Allium
This genus contains common and widely cultivated vegetable onion which is also known as bulb
onion or common onion. Other relative to this genus includes garlic, scallion, shallot, leek, chive
and Chinese onion. They are herbaceous biennial or perennial plant but usually considered as an
annual and harvested in its first growing season. Plants are about 30 cm long and a flowering
scape that can be up to 100 cm tall from an underground bulb.
Habit and Habitat: Plants are native to southwestern Asia. Now are growing all around the
world but mostly in temperate regions. Their original habitat is obscure; the plant probably arose
in Central Asia (Turkmenistan). They are not known to grown in wild.
V.S. of Allium leaf (Fig 11.8)
1. The tissue of leaf is clearly differentiated into epidermal, ground and vascular tissues.
2. The epidermis is uniserate and continuous and made up of small round cells with
cuticulrized outer walls.
3. Stomata are present in epidermis are slightly depressed.
4. Two types of mesophyll cells are present naming palisade and spongy parenchyma.
5. Internal to epidermis, two layers of columnar cells are present with abundant
chloroplasts. They are called palisade cells.
6. A few layers of isodiametric parenchyma spongy cells are present next to palisade cells.

7. The central Leaf part is hollow.

8. Vascular bundles are scattered in lower mesophyll regions, collateral and closed.
9. Each bundle is surrounded by parenchymatous bundle sheath.
Fig.11.8: V.S. of Allium leaf

11.3.3.3- Musa
The genus Musa includes bananas and plantains. There are 70 species of known Musa which are
used widely. They grow as high as trees. The banana and plantain plants are not woody. The
huge leaf stalks made their apparent stem. They are considered as gigantic herbs. Their many
species are used as food plants.
Habit and Habitat: They are tree-like herb with thick rhizome, pseudostem fleshy, succulent
formed by the imbricate leaf sheaths. The optimal temperature for fruit production is about 27°c,
and night time temperatures should not fall much below 18°c when the fruit is ripening or flavor
can be impaired. The optimum pH for the soil is 6-7.5.
V.S. of Musa leaf (Fig 11.9)
1. The epidermis is divided into upper and lower region.
2. The epidermal cells are compactly arranged, round in shape and have cuticularized outer
walls.
3. On the lower side of leaf surface, slight sunken stomata are present.

4. Both adaxial and abaxial thin-walled parenchyma cells are present next to epidermal
layers.
5. Cells in upper surface are larger in size than the lower surface.
6. The hypodermal layer is marked by the sub-epidermal cells.
7. These cells contain water but lacks chlorophyll.
8. Mesophyll layer is differentiated into palisade and spongy cells.
9. Palisade cell layers are present in upper 2-3 layers and consist of long columnar cells
with scanty intercellular spaces.
10. The spongy cells are smaller than palisade cells and are in isodiametric shape.
11. Numerous large air chambers are present at regular intervals towards the abaxial side.
12. The bundles are poorly developed, collateral and closed. They contain the scanty xylem.
13. Sclerenchyma patches are present in upper and lower sides of the vascular bundles.
Fig.11.9: V.S. of Musa leaf

11.3.3.4- Triticum
Wheat, a worldwide staple food, is the common and mostly grown crop of this genus. They are
basically grasses with economically important seeds as cereal grain. They were first cultivated in

the regions of the Fertile Crescent around 9600 BCE. The wheat kernel is called caryopsis is
considered as a type of fruit.
Habit and Habitat: The common habitats include fields, roadsides, areas along railroads, area
near grain elevators and open waste areas. They are known to grow in highly distributed areas
with exposed topsoil.
V.S. of Triticum leaf (Fig 11.10)
1. The epidermis constitutes the both upper and lower surfaces of the leaf.
2. They are composed of more or less oval cells and are covered by thick cuticle.
3. Some big, motor or bulliform cells are present in the upper epidermis surface.
4. Stomata with guard cells, pore and a stomatal chamber are present in both the epidermal
layers.
5. Mesophyll layer is not clearly differentiated in to palisade ad spongy parenchyma.
6. The cells adjacent to the epidermal layers are a bit longer but the cells of the central
mesophyll layer are oval and arranged in irregular fashion.
7. Many chloroplasts are present in the mesophyll cells.
8. Intercellular regions are present within the mesophyll layers.
9. Sub-stomatal chambers of the stomata are also placed in the region of mesophyll.
10. Many vascular bundles are present in the leaf and are arranged in a parallel series.
11. The vascular bundles present in center are the largest in size.
12. They are conjoint, collateral and closed.
13. All the bundles are surrounded by the double layered bundle sheath.
14. The bundle sheath outer layer consists of thin-walled cells whereas inner layer consists of
thick-walled cells.
15. The sclerenchyma patches are present on both the upper and lower surfaces of large
vascular bundles.
16. Xylem is present towards the upper surface while phloem is placed towards the lower
surface.
17. Xylem is constituted by vessels, tracheid and sometimes with the xylem parenchyma.
18. Phloem is composed of sieve tubes and companion cells.

Fig.11.10: V.S. of Triticum leaf

11.3.3.5- Oryza
This genus belongs to the grass family and includes the major food crop in all over the Asia,O.
sativa and O. glaberrima are most common species found. Plants are 1-2 m tall, wetland grasses
both the annual and perennial species. They are morphologically characterized by the single-
flowered spikelets whose glumes are completely suppressed. Species in this genus can be divided
by their genome’s types. Crossing is common in species of the same genome. The embryo rescue
technique is used for hybridizing different types.
Habit and Habitat:
As a crop, it's grown all over the tropical world. Europe, Africa, tropical and temperate Asia,
Australia, and North and South America are among the places where it is grown. Lowland
swampy environments are where they are discovered.
V.S. of Oryza leaf (Fig 11.11)
1. The upper epidermis constitutes the topmost layer or adaxial layer of the monocot leaf.
2. The cells are of cubic or barrel shape and are arranged compactly with no intercellular
spaces.
3. Thin cuticle covers the upper epidermal surface.

4. Large bulliform or motor cells are present on the upper epidermal layer of the cell.
5. Equal numbers of stomata are distributed among the both sides of epidermal layers i.e.,
amphistomatic distribution.
6. Air-cavities or sub-stomatal chambers are present above the stomata of the epidermal
layers.
7. The mesophyll tissue is not differentiated into palisade parenchyma and spongy
parenchyma.The mesophyll tissue is composed of 6-7 layers with large intercellular
spaces.
8. The spongy parenchyma cells are small oval or spherical or irregular with chlorophyll
and chloroplasts in them.
9. The vascular bundles are present in the mesophyll tissue.
10. Each bundle is consisting of xylem and phloem which is surrounded by the bundle
sheath.
11. Large barrel shaped endodermal cells compose the bundle sheath layer of the vascular
bundles.
12. These sheaths usually store starch granules and hence are called as starch sheath.
13. Xylem tissue is placed toward the upper epidermis of leaf.
14. It is consisting of xylem tracheid, xylem vessels, xylem parenchyma and xylem fibers.
15. Phloem is found towards the lower epidermal surface of the leaf.
16. Phloem is consisting of sieve tubes; sieve pores, companion cells, phloem parenchyma
and phloem fibers.
17. The bundles are conjoint, collateral and closed with endarch xylem.
18. A single layer lower epidermis is present below the undifferentiated mesophyll tissue on
the abaxial surface of the leaf.
19. The cells of this layer are cubical or barrel in space without the presence of intercellular
spaces.

Fig.11.11: V.S. of Oryza leaf

11.3.3.6- Zea
Zea belongs to the genus of flowering plants and the grass family. One of the most important
species of this genus is called Zea mays, which is also known as maize, corn or Indian corn.
Other wild species are commonly known as Teosintes. They are known to be native to
Mesoamerica.
Habit and Habitat: They are generally grown in sandy light, loamy medium and clay heavy
soils.Soil is needed to be well-drained to grow Zea. Slightly acidic or neutral soils are optimum
for some species like sweet corn. They do not grow in the shade. Nitrogen, potassium and
phosphorus are the important constituents of the soil for this crop to grow.
V.S. of Zea leaf (Fig 11.12)
1. The epidermis is present at both the upper and lower surfaces of the leaf.
2. Both surfaces contain stomata.
3. A thick cuticle covers the epidermal layers.
4. Cells in upper epidermis are large in size and are called bulliform cells or motor cells.
5. Mesophyll layer is present between the epidermal layers and they constitute the region of
chlorophyll containing cells.
6. Mesophyll layer is not differentiated into palisade and spongy parenchyma.

7. Mesophyll cells are spherical or angular and compactly arranged with few or no
intercellular spaces.
8. Numerous small and larger vascular bundles are present.
9. The bundles are collateral and closed.
10. All vascular bundles are covered by bundle sheath which is made up of parenchymatous
cells and contains starch and plastids.
11. Patches of sclerenchyma are present in the both ends of large vascular bundles, which are
extended up to the upper and lower epidermal layers.
12. Xylem and phloem are more visible in large bundles rather than small bundles.
13. Xylem is placed towards the upper epidermis whereas phloem is situated towards the
lower epidermis.
14. Xylem consists of tracheids, xylem vessels and xylem parenchyma.
15. Two large oral vessels represent metaxylem whereas a water cavity represents
protoxylem. Such water cavity is called lysigenous cavity.
16. Sieve tubes and companion cells constitute the phloem.
17. Less developed xylem and phloem surrounded by a bundle sheath is present in smaller
vascular bundles.
Fig.11.12: V.S. of Zealeaf

11.3.3.7- Bambusa
Bamboosare evergreen perennial grass plants. Their internodal regions of the stem are generally
hollow. Absence of secondary growth is observed in the stems of monocots, including the palms
and large bamboos, to be columnar rather than tapering. They are the fastest growing plants in
the world because of their unique property of rhizome-dependent system. They can grow up to
910 mm (36 inch) and hence are called Giant bamboos in general. They are very important in
culture and holds notable economic importance in the regions of South Asia, Southeast Asia and
East Asia. They are used as food, building materials, etc.
Habit and Habitat: Plants are native to all the continents excluding, Antarctica and Europe.
They are distributed from 47 S to 50 300 N and 4300 m up from the sea level. Genus is found in
temperate as well as tropical regions in association with a wide variety of mostly mesic to wet
forest types. Some species are adapted to grow in open grasslands or in specialized habitats.
V.S. of Bambusa leaf (Fig 11.13)
1. The upper and lower epidermal layers are uniserate.
2. The epidermal cells are more or less oval, large and empty bulliform cells.
3. Presence of stomata is observed in both the epidermal layers.
4. The mesophyll cells are not differentiated into palisade and spongy cells.
5. The mesophyll cells are compactly-arranged isodiametric cells with air-chambers in
between them.
6. Most of the vascular bundles are small but large bundles are present at some intervals.
7. The bundles are collateral and closed, arranged in parallel series.
8. Bundle sheath covers the small bundles which have xylem on upper side and phloem on
lower side.
9. The bundle sheath contains plastids with starch grains most often.
10. Xylem is constituted by trachery elements and phloem is made up of sieve tubes and
companion cells.
11. Sclernchyma cells are present in patches on both sides of the bundles.

Fig.11.13: V.S. of Bambusa leaf
11.4- SUMMARY
Vertical section of leaf provides the insight of the presence of epidermis as the outermost layer.
Cutin, a waxy substance coats the outermost surface, forming the layer of cuticle, especially on
the upper adaxial surface. The epidermal layer is consisting of stomata which carries out the
process of photosynthesis and helps in exchange of gases between the plant and environment.
Guard cells are also present in the epidermal layer. A compact bundle sheath with adaxial xylem
and adaxial phloem is also present. Trichomes, the epidermal appendages are present in the
surface which can either be unicellular or multicellular. Main vein is delineated from the spongy
mesophyll and a compact bundle sheath is present containing parenchymal cells. Adaxial xylem
and adaxial phloem is present in the enclosed area of bundle sheath. Some sclerenchymal cells
are also present in the vicinity of bundle sheath. A continuous network of vascular strand is
observed throughout the leaf and this network increases with finer dimensions when originated
from the main vein. The specialized parenchyma cells form the palisade mesophyll which
elongates in perpendicular direction to the leaf surface.
11.5- GLOSSARY
Adaxial- It is denoting the upper surface of a leaf).
Appendages-They are the projecting part of an invertebrate or other living organism, with a
distinct appearance or function.

Cosmopolitan-Something found all over the world.

Epiphytes-A plant that grows on another plant, especially one that is not parasitic.
Hemiepiphytes-It is a plant that spends part of its life cycle as an epiphyte.
Foliage- Collectively called for plant leaves.
Lamina-It is a thin, soft, pliable sheet or layer, especially of animal or vegetable tissue, serving
as a covering or lining, as for an organ or cell, e.g.- leaf lamina.
Petiole- The petiole is a stalk that attaches a leaf to the plant stem.
Photosynthesis-It is a process by which green plants and some other organisms use sunlight to
synthesize nutrients from carbon dioxide and water.
Precipitation-Precipitation is any product of the condensation of atmospheric water vapor that
falls under gravity from clouds.
Sessile- Attached directly by its base without a stalk or peduncle.
Transpiration- Transpiration is the process of water movement through a plant and its
evaporation from aerial parts, such as leaves, stems and flowers.
Trichomes- They are the small hair or other outgrowth from the epidermis of a plant, typically
unicellular and glandular.
Vines- a climbing or trailing woody-stemmed plant

1. Cuticle is present on the _________ surface of leaf.
2. The epidermal appendages present on the surface of leaf are called_____________.
3. __________________ causes the delineation of main vein on the leaf.
4. _____________ cells elongate in the perpendicular direction of leaf surface.
5. _____________ are the epiphytes and hemiepiphytes and commonly called
as_____________.
6. _____________ genus is known for manufacturing high-quality woodwind instruments
and furniture.
7. ______________ crystal is present in the mesophyll tissue of Eucalyptus.
8. ______________ species of Cycas is endemic inIndia.
9. Eucalyptus sepals and petals are fused together to form cup-like structure over the
stamens, called ___________.
10. Cycas stomata are ____________.
11. ______________are considered as gigantic herbs.

12. Ficus mesophyll cells can be _________and ________.

13. Stomata inNerium is placed in the pit and protected by ______________.
14. Allium is grown in ___________ regions.
15. _______________are evergreen perennial flowering plants.
16. In Zea cells in upper epidermis are large in size and are called ____________ or
___________.
ANSWERS TO SELF ASSESSMENT QUESTIONS
1. Adaxial
2. Trichomes
3. Mesophyll cells
4. Palisade mesophyll
5. Ficus, Figs
6. Pyrus
7. Calcium oxalate
8. C. circinalis
9. Operculum
10. Haplocheilic
11. Musa
12. Bifacil, Isobilateral
13. Trichomes
14. Temperate
15. Bambusa
16. Bulliform cells, Motor cells
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3. Haupt, A. W. (1953). Plant morphology (No. 581.4 H38).
4. Mauseth, J. D. (2014). Botany: an introduction to plant biology. Jones & Bartlett
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(https://patents.google.com/patent/USPP26619).
11. Sunset Books. (1995). Sunset Western garden book. Sunset Publishing Company.
12. List, P. (2015). The Plant List. Version 1.1.
13. Group, I. S. I. S. S. The Global Invasive Species Database (GISD) and international
information exchange: using global expertise to help in the fight against invasive alien
species.
14. Sheikh, M. I. (1993). Trees of pakistan (Vol. 110). Islamabad: Pictorial Printers.
15. Krause, S., Hammer, K., & Buerkert, A. (2007). Morphological biodiversity and local use
of the Himalayan pear (Pyrus pashia) in Central Bhutan. Genetic Resources and Crop
Evolution, 54(6), 1245-1254.
16. ZAMANI, A., ATAR, F., & JOUHARCHI, M. (2009). Pyrus pashia (Rosaceae), a new
record for the flora of Iran.
17. Sellers, C. H. (1910). Eucalyptus: its history, growth, and utilization. AJ Johnston, 13.
18. https://www.indiastudychannel.com/resources/168696-Anatomy-or-internal-structure-of-
a-Monocotleaf.aspx#:~:text=In%20monocot%20leaf%20mesophyll%20tissue, cells%20
with%20chloroplasts%20and%20chlorophyll.
19. Shakir, H. M., & Baji, S. H. (2016). Anatomical study of some characters in certain
species of genus Ficus L. growing in Iraq. J Biol Agric Healthcare, 6(12), 98-105.
20. https://www.studyadda.com/notes/11th-class/biology/plant-kingdom/some-
representative-rymnosperms/9569
21. https://brainly.in/question/43171596

11.8- SUGGESTIVE READINGS

1. https://biologyreader.com/difference-between-dicot-and-monocot-leaf.html#Anatomy
2. https://www.biologydiscussion.com.
3. https://www.microscopemaster.com/leaf-structure-under-the-microscope.html
4. https://garden.org/onlinecourse/Act7.htm.

1. What are the characteristics of a Nerium leaf when observed under microscope?
2. Draw a well labeled diagram of vertical section of Eucalyptus leaf.
3. Explain the Pine leaf anatomy.
4. Give an insight on Pyrus leaf.
UNIT-12 STUDY OF EPIDERMAL PEELS OF LEAVES,

PREPARATION OF STOMATAL INDEX AND
DEMONSTRATION OF THE EFFECT OF ABA ON
STOMATAL CLOSURE
12.1- Objectives

12.2- Introduction
12.3- Study of epidermal peels of leaves to study the structure of stomata
12.3.1- Monocot species
12.3.1.1- Triticum
12.3.1.2-Zea
12.3.1.3- Tradescantia
12.3.1.4- Saccharum
12.3.1.5- Cyanodon
12.3.1.6- Musa
12.3.2- Dicot species
12.3.2.1-Sedum
12.3.2.2- Pelargonium
12.3.2.3- Abelmoschus
12.3.2.4- Hibiscus
12.4- Preparation of stomatal index
12.4.1- Triticum
12.4.2-Carex
12.4.3- Avena
12.4.4-Gladiolus
12.4.5-Juncus
12.4.6-Allium
12.4.7-Orchis
12.5- Demonstration of the effect of ABA on stomatal closure
12.6- Summary
12.7- Glossary
12.8- Self Assessment Questions
12.9- References

12.1 OBJECTIVES
After reading this unit students will be able -
1. To study the epidermal peels of leaves in order to understand the development and final
structure of stomata.
2. To prepare stomatal index and demonstrate the effect of ABA on stomatal closure

12.2 INTRODUCTION
The epidermis is the outermost layer which covers the cell surface. It protects the inner tissues
from adverse natural disasters like high temperature, desiccation, mechanical injury, external
infection etc. The epidermis of monocotyledonous plant is originated from the periblem along
with the cortex.
Stomata of monocots are tiny and are present in upper and lower epidermis of monocot leaves.
They are generally surrounded by dumbbell-shaped guard cells. They are arranged in regular
arrays. They are usually equally distributed in both the upper and lower epidermis. The stomata
distribution in monocots is generally known as amphistomatic distribution. The frequency of
transpiration can be higher than dicot leaf due to the presence of amphistomatic distribution of
stomata.
Stomata of dicots are either present on the lower side of the leaf or they might be only present
only on the lower side of the leaf. Such leaves having stomata present on the lower side of the
leaf are given with a term, hypostomatous leaves. This special arrangement called
hypostomatous distribution of stomata allows water to be conserved in the leaves.
The stomatal index is the measurement of number of stomata presents to the total number of
epidermal cells, each stoma being counted as one cell in the form of percentage. The stomatal
index (I) is defined as average number of stomata cells present per square millimeter of the
epidermis of the leaf. The formula for calculating the stomatal index (I) is given by the formula
or equation, I = S / E + S; where I, stomatal index; S, number of stomata cells per unit area; and
E, number of epidermal cells per unit area.
12.3 STUDY OF EPIDERMAL PEELS OF LEAVES TO

UNDERSTAND THE DEVELOPMENT AND FINAL
STRUCTURE OF STOMATA
METHODOLOGY
Materials
 Plants with suitable leaves with smooth surface.
 Compound microscope with magnification up to 400x
 Forceps and blades
 Microscope slide
 Cover slips

 Cotton, Tissue paper

 Alcohol or ethanol
 Digital camera to capture microscopic images of structure of epidermal peel and stomata
Procedure (Fig 12.1)
a) Collect clean leaves with smooth surface.
b) Make a clean cut at one end of the leaf structure with the help of blades.
c) Clean the microscopic slides using ethanol. Place the specimen on the microscopic slide.
d) Clamp down the thick spot including the portion of epidermis along the cut using fine
forceps.
e) Use forceps to pull the various layers of tissues.
f) At first thick wad of tissues will be visible.
g) After pulling sub-epidermal layers, epidermis layer is observed.
h) Place it faces up on microscopic slide and crops it so that some of the thin strip is
retained.
i) Add water and place the cover slip.
j) Observe slide under the microscope.
Fig.12.1: Preparation of epidermal peel.

12.3.1- Monocot species

12.3.1.1 Triticum
Wheat is a worldwide staple food and is the common member of this genus. It basically belongs
to the grasses which has economically important seeds as cereal grain. They were first cultivated
in the regions of the Fertile Crescent around 9600 BCE. The wheat kernel is called caryopsis
considered as a type of fruit.
Botany of the plant
Triticum is a cultivated annual crop with herbaceous, erect, cylindrical and fistular stem. The
unbranched glabrous stem comprised of distinct node and internodes and root is adventitious.
Hermaphrodite, incomplete and zygomorphic flower lies between superior and inferior palea.
Morphology of leaf
Leaf is simple, alternate and green. No stipule is present and hence considered as exstipulate.
Leaf has entire margin;acute apex and it has sheathing leaf base. Ligule is present at the junction
of leaf-sheath and leaf-blade membranous ligule present. Parallel venation is observed.
The epidermal peel of Triticum leaf (Fig 12.2)
1. The upper and lower epidermal surfaces are present on the leaf surface.
2. Both the epidermal layers are uniseriate.
3. The epidermal layers are composed of more or less oval cells.
4. Some big, motor or bulliform cells are present in the epidermal surface.
5. Stomata are present on both the epidermal layers and they are consisting of a pore, guard
cells and a stomatal chamber.A thick cuticle covers the outer walls of the epidermal cells.
6. The epidermal cells are thin walled, hyaline and have large vacuole.
7. The bulliform cells present in the middle are the tallest and the sides of the other cells
present on sides are smaller.
8. The epidermal cells contain abundant water but are devoid of chloroplastids.
9. Dumb-bell shaped stomata are found.

Fig.12.2: The structure of leaf epidermis of Triticum.
12.3.1.2- Zea
Zea belongs to the grass family. One of the most important species of this genus isZea mays,
which is also known as maize, corn or Indian corn. Other wild species are commonly known as
Teosintes. They are known to be native to Mesoamerica.
Botany of the plant
Zea is an annual cultivated crop with erect, cylindrical, herbaceous and solid stem. Smooth stem
is defined in distinct notes and internodes. Adventitious stilt roots are present. Plant is
monoecious.
Morphology of leaf
Leaf is simple, alternate and linear. Long ligule is present. Venation is multicostate parallel.
The epidermal peel of Zea leaf (Fig 12.3)
1. The single-layered epidermis is present in both upper and lower surfaces of the leaf.
2. On the both epidermal surfaces, stomata are present.
3. Both epidermal layers are covered by thick cuticle on outer surface.
4. Some cells are larger in the upper epidermal layer and are called bulliform cells or motor
cells.
5. The guard cells on the stomata are peculiarly dumb-bell-shaped in form.

6. The subsidiary cells lie adjacent to stoma mother cell and they occur on 2 sides of the
guard cells.
7. Such stomata are also referred as Zea type.
Fig. 12.3: The epidermal peel of Zea leaf

12.3.1.3- Tradescantia
This genus includes 75 species of herbaceous perennial wildflowers. They are native to the New
World form southern Canada to northern Argentina, including West Indies. Some members of
this genus are popularly known by the names such as spiderwort or Indian paint. They are used
as ornamental plants in many parts of the world. They are weakly-upright to scrambling plants.
They can grow up to 30-60 cm in height.
Botany of the plant
They are herbaceous annual orperennials. They have adventitious roots. Stem is like rhizome,
branched jointed with swollen nodes.
Morphology of leaf
Leaves are simple, alternate with sheathing base and narrow grass-like blades. Leaves have
entire margin. They are linear, oval or lanceolate parallel venation.
The epidermal peel of Tradescantia leaf (Fig 12.4)
1. The epidermal cells are arranged irregularly.
2. There are no intercellular spaces present between the epidermal cells.
3. Numerous small stomata are scattered in epidermal cells.

4. Each stomatal pore is guarded by 2 bean-shaped cells.

5. The guard cells contain chloroplasts and a nucleus.
6. The inner boundary of guard cell is concave and thick whereas the outer boundary is thin.
7. Both open and close stomata are present which are regulated by guard cells.
8. 4 subsidiary cells surround the stoma mother cell.
Fig.12.4: The epidermal peel of Tradescantia leaf

12.3.1.4- Saccharum
The species belonging to this genus include the tall perennial plants of the broomsedge tribe in
the grass family. They are found in all around the tropical, subtropical and warm temperate
regions of Africa, Eurasia, Australia, Americas and assorted oceanic islands. Sugarcanes are the
common members of this genus and also it includes Ravenna grass which is used as ornamental
purposes.
Botany of the plant
They are large tropical grasses with multiple stems or culms each containing series of nodes
separated by internodes. The internode length can grow up to 30 cm; consists of sucrose which is
stored in parenchyma cells and vascular tissue. The stem is made up of parenchyma cells and is
not hollow like other grasses. Mature stems have leaf spindles made up by enclosing the number
of leaves to form the structure. Buttress roots are developed in the plants of this genus to anchor

the plant. The roots penetrate and grow downwards which allows sufficient water absorption
under the water or drought stress.
Morphology of leaf
The abaxial (under) surface of leaf blade is pubescent i.e. hairy and the adaxial (top) surface of
the leaf is glabrous i.e. without hairs. The leaf blade terminatesin a pointed tip. The length and
width of leaf blade varies from 60-150 cm long and 2-10 cm, respectively. The leaves are
attached at the node from the stem and then wraps the stem to form a sheath enclosing internode
from which node subtends.
The epidermal peel of Saccharum leaf (Fig 12.5 A,B)
1. The outermost layer is the single layered epidermis.
2. The epidermis is covered by the cuticle from outside.
3. The bulliform or motor cells are present on the epidermal layers.
4. The epidermal layer is interrupted by presence of the stomata.
5. The stomata are guarded by the guard cells.
6. The guard cells contain chloroplasts and a nucleus.
7. The guard cells regulate the opening and closing of stomata.
Fig.12.5 (A): The stomatal and epidermal structure at abaxial surface of Saccharum.

Fig.12.5 (B): The stomatal and epidermal structure at adaxial surface of Saccharum
12.3.1.5- Cynodon
This genus belongs to the large and ubiquitous family of monocotyledonous flowering plants
which are known as grasses. There are around 780 genera and 12000 species of the Poaceae
family. They have economic importance as they provide staple foods from cereal crops like
maize, wheat, rice, barley and millets. All have hollow stems except at the nodes. The species of
Cynodonare commonly called as Bermuda grass or dog tooth’s grass. Many species of this genus
have originated in southeast Africa. They are also known as ubiquitous cosmopolitan weed.
Botany of the plant
Cynodon term comes from greek word meaning ‘dog-tooth’. It is a genus belonging to grass
family. They best thrive in warm temperate to tropical regions. The stems are slightly flattened
with a tingling of purple color in them. The stem is erect and can grow up to 1-30 cm. Deep roots
growing up to 2 meters, allows the resistance and tolerance during drought and water stress with
effective ability of water absorption.
Morphology of the leaf
The short leaf blades are grey-green in color. Their length varies from 2-15 cm with rough edges.
The epidermal peel of Cynodon leaf (Fig 12.6)
1. Large, highly vacuolated and thin-walled cells are present in epidermis.
2. They are called bulliform i.e., bubble-like cells.
3. They are present on both surfaces of the leaf but mostly on the upper surface of leaf.
4. The cells contain water but no chlorophyll.

5. They have thin wall but the other wall is thick and cutinized mostly by silica.
6. Stomatal pore is covered by guard cells.
7. Subsidiary cells surround guard cell.
Fig.12.6: The epidermal peel of Cynodon leaf
12.3.1.6- Musa
The genus Musa includes bananas and plantains. There are 70 species of known Musa which are
used widely. They grow as high as trees. The banana and plantain plants are not woody. The
huge leaf stalks made their apparent stem. They are considered as gigantic herbs. Their many
species are used as food plants.
Botany of the plant
The plant species are perennial, tall, tree like herbs. The stem is underground, rhizomatous,
perennating sheathing leaf bases rolled upon one another to form a pseudoaerial stem. Roots are
adventitious.


The leaves are radical, simple, alternate, exstipulate, large and broadly elliptical. The leaf base
may grow up to 6 feet in length. Unicostate parallel venation is observed.
The epidermal peel of Musa leaf (Fig 12.7)
14. The epidermis is divided into upper and lower regions.
15. The epidermal cells are compactly arranged and round in shape and have cuticularized
outer walls.
16. On the lower side of leaf surface, slight sunken stomata are present.
17. Both adaxial and abaxial thin-walled parenchyma cells are present next to epidermal
layers.
18. Cells in upper surface are larger in size than the lower surface and they are markedly
different from the mesophyll.
19. The stomata are Rheo type with two lateral and two-three polar cells and four subsidiary
cells and are sunken.
Fig.12.7: The epidermal peel of Musa leaf

12.3.2 Dicots species
12.3.2.1- Sedum
It is the genus of the family Crassulaceae. The genus is commonly called as stonecrop. They are
succulent perennial herbs or shrubs. They plants are primarily found in the Northern Hemisphere
but also found in some parts of Southern Hemisphere like in Africa and South America.

Botany of the plant

Sedumis an annual, biennial and perennial herb. The plant possesses succulent leaves and stems.
The plants may vary from creeping herbs to shrubs. The flowers usually have five petals, seldom
four or six.
Leaves are simple or compound, opposite, alternate or whorled, exstipulate, more or less thick
and fleshy. The leaves are fleshy modified to store the water.
The epidermal peel of Sedum leaf (Fig 12.8)
1. Stomata are ofanisocytic cells
2. Usually, six subsidiary cells are possessed around the stomata of Sedum.
3. Two guard cells are formed after the spiral series of cell division from subsidiary cells.
4. Subsidiary cells act as spacers in order to give ordered arrangement of stomata.
Fig.12.8: Structure of stomata and epidermis anatomy of Sedum.

12.3.2.2- Pelargonium
The plants belonging to this genus are evergreen perennials, succulents and shrubs. They are
more commonly known as geraniums, pelargoniums, or storksbills. The plants thrive in warm
temperate and tropical regions throughout the world and many species are found in southern
Africa. The plants are known to be tolerant of drought, heat and minor frosts.
Botany of the plant
Species belonging to this genus are herbaceous annuals, shrubs, subshurbs, stem succulents and
geophytes. Stem is erect occasionally branched which on flowering bears five-petaled flowers in

umbel-like clusters and are zygomorphic.Stem is fleshy and is found to be thick below and often
woody.
Leaves are alternate, incised or palmately lobed or pinnate or incised up to the base or compound
often on long stalks. The leaves are rarely entire and stipulate. Some light or dark patterns are
observed on the leaves. The leaves have thick cuticle to resist drought stress.
The epidermal peel of Pelargonium leaf (Fig 12.9)
1. Stomata are orderly arranged on the lower surface of the epidermis.
2. Stomata are of amonocytic type and their size is 4mm long when measured by the length
of central vein.
3. The space is enlarged to become substomatal cavity due to the presence of stoma.
Fig.12.9: Structure of stomata and epidermal peel of Pelargonium leaf.

12.3.2.3- Abelmoschus
Abelmoschus name has derived from Abrabian language, meaning ‘father of musk’ or ‘source of
musk’ related to the scented seeds. The genus belongs to the Malvaceae. It is are grown in
humid, temperate and wet regions. Formerly it was included under Hibiscus genus. They are
native to tropical Africa, Asia and northern Australia.

Botany of the plant

Species belonging to this genus comprise annual and perennial herbaceous plants. The plant
height goes up to 2 m tall. It is a perennial shrub.
Leaf is ovate to pentagonal in shape, palmately lobed with 3-7 lobes. 5-6 bracts measuring 7-11
mm in length are present. The leaves are 10-40 cm long and broad.
The epidermal peel of Abelmoschus leaf (Fig 12.10 A,B,C)
1. Epidermal cells vary from polygonal to irregular in shapes and anticlinal walls are wavy
on adaxial surface.
2. Epidermal cells are irregular and anticlinal wall of wavy and undulating on abaxial
surface.
3. Average number of epidermal cells is 17.
4. Average number of stomata present on adaxial and abaxial surfaces are8 and 6
respectively.
5. Stomata are anomocytic, amphistomatic and often anisocytic.
6. Large irregular cells in the form of mucilaginous are present on both surfaces.
Fig.12.10 (A): The structure of polygonal epidermal cells in Abelmoschus leaf.

Fig.12.10 (B): The structure of isodiametric epidermal cells in Abelmoschus leaf.
Fig.12.10 (C): The structure of irregular epidermal cells in Abelmoschus leaf.
12.3.2.4- Hibiscus
This is another genus of mallow family named as Malvaceae. Numerous species numbering to
several hundred comprised of this genus. Species belonging to this genus thrive in warm
temperate, subtropical and tropical regions throughout the world.
Botany of the plant

The plants belonging to this genus are used as ornamental shrubs usually cultivated in tropics.
These are perennial. Stems are erect, branched, cylindrical, solid, woody and glabrous. Roots are
tap, branched and deep. Inflorescence is solitary axillary.
Leaves are alternate, simple and possess petiole. Leaf has stipule and serrate margin. Unicostate
reticulae venation is present. Leaf is ovate and glabrous.
The epidermal peel of Hibiscus leaf (Fig 12.11)
1. Epidermal cells vary from polygonal to irregular in shapes and anticlinal walls are
straight on adaxial surface.
2. Epidermal cells are irregular and anticlinal wall of wavy and undulating on abaxial
surface.
3. Average numbers of epidermal cells are 22.
4. Average number of stomata present on adaxial and abaxial surfaces are8 and 6
respectively.
5. The vascular bundles are present in a ring arc form.
Fig.12.11: The structure of stomata and epidermis in Hibiscus leaf.
12.4- PREPARATION OF STOMATAL INDEX

The stomatal index is defined as the number of stomata presents to the total number of epidermal
cells; each stoma being counted as one cell in the form of percentage. The stomatal index (I) is
the measure in terms of an average number of stomata cells present per square millimeter of the
epidermis of the leaf. The formula for calculating the stomatal index (I) is given by
I=S/E+S

where,
I= Stomatal index
S= number of stomata cells per unit area
E= number of epidermal cells per unit area
Stomatal Distribution
Stomatal distribution refers to the distribution of stomata among monocot and dicot
plants.Stomata are found on the plant surfaces like leaves, stem, etc.
Methodology
Materials required
 Compound microscope
 Microscope slides
 Cover glasses
 Forceps
 Spirit lamp
 Small watch glass
 Blade
 Cello tape
 Microscopic grid
 Dark colored pencil with sharp lead
 Chloral hydrate solution
Procedure
Preparation of lamina (Fig 12.12)
A mature leaf is taken either small or if big then cut it up to 5 mm square pieces from the middle
portion between the lamina and the midrib.
Fresh leaf
a) Separate the epidermis or peeled off in thick leaves through breaking into pieces.
b) After separating the epidermis, treat with chloral hydrate.
c) Boil the leaf or leaf pieces in chloral hydrate in a test tube placed in water bath. It will
allow epidermis to be separated.
d) Now place the peeled epidermis on a slide with 1-2 drops of chloral hydrate or glycerin
as mounting medium.

e) Let the content be cool and then place a cover glass and then observe it under
microscope.
f) Prepare separate slides for upper and lower epidermis.
Dry leaf
a) Add dry leaf in a test tube with chloral hydrate and heat it on water bath for 30 min.
b) Cut the leaf into two pieces and observe them under microscope for observing stomata.
c) Veins should be faced downwards through putting the cleared half on the microscopic
slide and then upper epidermis is visible.
d) Veins facing upwards should be through putting the other half of the leaf on the
microscopic slide which allows the lower epidermis to be visible.
e) Now peeled off the epidermis from both adaxial and abaxial surfaces (You will get now
upper and lower epidermis)
f) Add two drops of glycerin to the cut pieces of epidermis on the slide and place a cover
glass over it.
g) Observe them under microscope to trace the epidermal cells and stomata.
Tracing of cells
a) Draw an 8-10 cm square on a drawing sheet or any unit area.
b) Place the specimen after treatment in slide under the microscope.
c) Focus epidermal cells and stomata using microscope to magnify significantly.
d) Trace epidermal cells and stomata using camera lucida in the square and draw the figure
with help of square grid.
e) Count the epidermal cells and stomata within the boundaries of drawn square.
To determine the stomatal index of any plant leaf:
1. Leaf pieces are cleared and mountedbetween the margin and midrib.
2. The lower surface is examined with an objective piece of microscope.
3. The sample can be observed by the eyepiece.
4. The number of epidermal cells and number of stomata are counted within a square grid.
5. The stomatal index is determined on both the leaf surfaces.

Separate epidermal
peel and break it into
pieces
Take small lamina

or cut it into pieces
Add the leaf in Dried lamina

Fresh lamina test tube and treat
it with chloral
hydrate Add dried leaf in a
test tube filled of
chloral hydrate
Boil the contents Chloral hydrate
within the test tube Chloral hydrate
Heat for 30
minutes and then
cut the leaf into
two pieces
Place the epidermis on the slide and

add 1-2 drops of chloral hydrate Observe under the microscope
Fig.12.12: Procedure of preparing lamina for the study of stomata counting through the
observance under the microscope.
Generally, on the ventral and dorsal surfaces, numbers of stomata found are 20-23 and 13-16,
respectively.
Stomatal index of Triticum:
The stomatal frequencies vary on both the adaxial and abaxial surfaces of the leaf. It can also
vary in the presence of stress. The stomatal frequency on adaxial surface of leaf is 2.77 whereas
stomatal frequency on abaxial leaf surface is 2.35mm2. The ratio of stomatal index on abaxial to
adaxial surface is 0.78 and 0.81.
Stomatal index of Carex:
The stomata are only present in the abaxial surface. Stomata are absent on the leaf margins. The
highest frequency of stomata found in Carex nigra is 179 mm-2 and the lowest frequency of

stomata is found in C. distans is 120 mm-2. The stomata are of paracytic type. The length of
guard cell is in between 27.06-36.3 µm.
Stomatal index of Avena:
The stomata are generally found on the both surfaces of the leaf. The density of stomata on
adaxial surface is higher than the abaxial surfaces but this situation is reverse in Avenaeriantha.
Stomata are usually found in the regular rows of the lamina. The highest density of stomata is
found in A. textilis by 200 mm-2 and the lowest density of stomata are found in A. arundinaceus
by 43 mm-2. The guard cells are large with average cell length of 55.86 µm on the adaxial
surfaces and 30.96 µm on the abaxial surfaces.
Stomatal index of Gladiolus:
The stomata are found on both adaxial and abaxial surfaces of the leaf. The density is higher in
the abaxial surface with 110 mm-2 and lower in adaxial surface with stomatal density of 18.69
mm-2. The stomata are not arranged with regularity in whole leaves. The stomatal type found in
Gladiolus is anomocytic. The length of abaxial guard cell is 33.17 µm in average in Gladiolus.
Higher the stomatal frequencies, longer are the guard cells in length.
Stomatal index of Juncus:
The stomata are found on abaxial surfaces only. They are regularly situated in linear groups. The
stoma is of anomocytic type. The stomatal density is 35-83 mm -2. The length of guard cells of
Juncus is in between 21.6-35.3 µm.
Stomatal index of Allium:
The stomata are present on both the surfaces. The Allium has greatest stomatal frequencies with
an abaxial average density of 143 mm-2. The stomatal frequencies onabaxial surfaces are greater
than those of adaxial surfaces of the leaf. The largest guard cells are always found in the abaxial
surface of the leaf. The size of stomata is equal on both the surfaces i.e. 32 µm and the length of
guard cells vary from 23.64-39.48 µm.
Stomatal index of Orchis:
The stomata are only found in the abaxial surfaces of leaf. These are scattered or arranged
without any regularity on the whole leaf. The stomata are of anomocytic type. The average
length of Orchisguard cell is 67.4 µm. The stomatal density is 78 mm-2 for Orchis.

Table 1: Types of Stomata, Number of stomata and Stomata index of Different Species
Name of Type of Number Abaxia Adaxial Number Number Stomatal index

Species stomata of l surface of of (Abaxial/Adaxi
stomata surface epiderma epidermal al)
per unit Density l cells in cells in
area(m Density (mm-2) abaxial adaxial
m2) (mm-2) surface surface
Triticum ** 9859 ** ** 5170 7490 0.78/0.81

aestivum
Carexhirt Paracytic ** 245 144 ** ** 16.2-23.8

a
Avena Potamoget ** 65 44 27-35 25-48 **

sativa on
Gladiolus Anomocyti ** 110 18.69 ** ** **

c
Juncus Anomocyti ** ** ** ** ** **
c
Allium ** ** 143 ** ** ** **
Oryza ** ** ** ** ** ** **
sativa
Zea mays Paracytic ** 100±19 71±15 68-158 52-94 13.2/16.9 (0.71)
Saccharu ** ** ** ** 176-351 59-167 **

n
officinaru
m
** Data was not available
12.5- DEMONSTRATION OF THE EFFECT OF ABA ON

STOMATAL CLOSURE
Abscisic acid i.e., ABA is a plant hormone which is involved in plant response towards reduced
water availability. It causes reduction in guard cell turgor pressure in its absence by closing the
stomatal pore aperture. This is how water is conserved during periods of drought. The cytosolic

Ca2+ mediates the reduction in turgor of guard cell through a signal transduction pathway which
is elicited by ABA. ABA is responsible for Ca 2+ mobilization pathway which involves cyclic
adenosine 5’- dephosphoribose (cADPR). The cADPR microinjections in guard cells causes
reduction in turgor that was preceded by increase concentration of free Ca2+ in the cytosol.
The effect of ABA on stomatal closure (Fig 12.13)
1. ABA controls the stomatal closure response in various environmental stresses.
2. During the period of drought or reduced water, ABA is abundant in leaves.
3. It promotes the reduction in stomatal aperture in order to reduce the extent of
transpirational water loss.
4. The reduction in stomatal pore width is caused by decrease in the turgor of the two guard
cells surrounding the stomatal pore.
5. ABA promotes the efflux of potassium salts from the guard cells.
6. Such operation of ABA takes place through Ca2+ dependent signal transduction pathways.
7. ABA increases the cytosolic free Ca2+ concentration.
8. A large number of Ca2+ permeable channels are present on both the plasma membrane
and the endomembranes which are involved in the specific signal transduction pathways.
9. This Ca2+ releasing channelshave the dynamic properties which produce the stimulus
response by which opening and closing of stomata is controlled.

Fig.12.13: Diagrammatic representation of (A) ABA mediates stomatal closing and (B) ABA
inhibits stomatal opening in guard cells.
12.6- SUMMARY
The epidermis is the outermost layer of the cell surface which protects the inner tissues from
adverse natural disasters like high temperature, desiccation, and mechanical injury, external
infection etc. the tiny stomata are present in upper and lower epidermis of leaves, generally
surrounded by dumbbell-shaped guard cells. These stomata are arranged in regular arrays and
areequally distributed in both the upper and lower epidermis. The stomatal index is the
measurement of number of stomata forms to the total number of epidermal cells, each stoma
being counted as one cell in the form of percentage. Abscisic acid (ABA) is plant hormone
involved in plant response to reduced water availability. It does so by reduction in guard cell
turgor pressure and thus closing the stomatal pore aperture.
12.7- GLOSSARY
Abaxial: Facing away from the stem of a plant or to the lower surface of a leaf.
Adaxial: Facing towards the stem of a plant or to the upper surface of a leaf.
Amphistomatic: It represents having stomata on both surfaces.

Anomocytic: It represents stomata have guard cells that are surrounded by cells that have the
same size, shape and arrangement as the rest of the epidermis cells.
Cosmopolitan: Found all over the world.
Deciduous: Shedding its leaves annually.
Desiccation: The removal of moisture from something.
Gigantic: It means great size or extent; huge or enormous.
Herbaceous: It denotes to herbs.
Hyaline: Glassy and translucent in appearance.
Imbricate: Adjacent edges overlapping.
Obscure: Uncertain not unknown.
Pantropical: Occurring or distributed throughout the tropical regions of the earth.
Perennial: Living for several years.
Periblem: It is a primary meristem that gives rise to the cortex and is located between plerome
and dermatogen.
Prairies: A large open area of grassland.
Scrambling: It is having a stem too weak to support itself, instead attaching to and relying on
the stems or trunks of stronger plants.
Staple: They are regularly consumed in large quantities as to form the basis of a traditional diet
and which serves as a major source of energy and nutrients.
Transpiration: It is the process of water movement through a plant and its evaporation from
aerial parts, such as leaves
Turgor: The force within the cell that pushes the plasma membrane against the cell wall.
Ubiquitous: Found or existing everywhere.
Uniseriate: Arranged in a single row, layer, or series.

1. The wheat kernel is called __________.
2. The stomatal type found in Gladiolus is ______________.
3. The species of Cynodonare commonly called as ___________.
4. Carex has _________ type of stomata.
5. ABA is responsible for Ca2+ mobilization pathway which involves _____________.

6. The Orchisstomata are of ___________ type.

7. _________ are commonly called as Rushes.
8. The ratio ofTriticum stomatal index on abaxial to adaxial surface is _______ and
__________.
9. The stomata of maize are also referred as ___________.
10. ____________ are herbaceous perennial.
ANSWERS TO SELF ASSESSMENT QUESTIONS
1. Caryopsis
2. Anomocytic
3. Bermuda grass
4. Paracytic
5. cyclic adenosine 5’- dephosphoribose (cADPR)
6. Anomocytic
7. Juncus
8. 0.78, 0.81
9. Zea type
10. Tradescantia
12.9- REFERENCES
1. Allen, G. J., & Sanders, D. (1997). Vacuolar ion channels of higher plants. In Advances
in Botanical Research, 25: 217-252). Academic Press.
2. Aminian, R., Mohammadi, S., Hoshmand, S. A., & Khodambashi, M. (2010). The
genetic analysis of stomatal frequency and size, stomatal conductance, photosynthetic
rate and yield in wheat (Triticum aestivum L.) using substitution lines series. Wheat
Information Service, 110: 25-34.
3. Ball, P. W., & Reznicek, A. A. (2002). Carex Linnaeus, Sp. P1. 2: 972. 1753. Flora of
North America Editorial Committee.
4. Christmann, A., Hoffmann, T., Teplova, I., Grill, E., & Müller, A. (2005). Generation of
active pools of abscisic acid revealed by in vivo imaging of water-stressed
Arabidopsis. Plant physiology, 137(1), 209-219.

5. Yakandawala, D. M. D., Sirisena, U. M., & Dassanayake, M. D. (2005). Two New

Records of Juncus Species (Rush Family-Juncaceae) In Sri Lanka. Ceylon Journal of
Science (Biological Science), 33, 67-76.
6. Goldblatt, P., & Manning, J. (1998). Gladiolus in southern Africa. Fernwood Press.
7. Goldblatt, P.; De Vos, M. P. (1989). "The reduction of Oenostachys,
Homoglossum and Anomalesia, putative sunbird pollinated genera, in Gladiolus L.
(Iridaceae-Ixioideae)". Bulletin du Muséum National d'Histoire Naturelle, Section
B. 11 (4): 417–428.
8. Hipp, A. L. (2007). Nonuniform processes of chromosome evolution in sedges (Carex:
Cyperaceae). Evolution: International Journal of Organic Evolution, 61(9), 2175-2194.
9. http://agropedia.iitk.ac.in/content/gladiolus-swordlily#:~:text=It%20has%20its%20
natural%20habitat,of%20flowers%20known%20as%20spikes.
10. https://amrita.olabs.edu.in/?sub=79&brch=7&sim=128&cnt=1
11. https://byjus.com/biology/preparing-a-temporary-mount-of-a-leaf-peel-to-show-stomata/
12. https://www.biologydiscussion.com/experiments/experiment-to-observe-temporary-
mount-of-a-leaf-peel-to-show-stomata/1733
13. https://www.biologydiscussion.com/plant-tissues/the-epidermal-tissue-system-of-plants-
with-diagrams/13880
14. https://www.cabi.org/isc/datasheet/115039#:~:text=J.,Hemisphere%20as%20an
%20ornamental%20plant.
15. https://www.illinoiswildflowers.info/grasses/plants/oats.html#:~:text=Habitats
%20consist%20of%20cropland%2C%20abandoned,grain%2C%20forage%2C%20and
%20straw.
16. https://www.rainforest-alliance.org/species/orchid#:~:text=Most%20orchid%20species
%20grow%20in,lie%20along%20the%20Andes%20Mountains.
17. https://www.ukessays.com/essays/biology/determination-of-stomatal-index-biology-
essay.php
18. Jermy, A. C., Simpson, D. C., Foley, M. J. Y., & Porter, M. S. (2007). General structure
of Cyperaceae. Sedges of the British Isles. Sedges of the British Isles. BSBI Handbook,
(1), 2-26.

19. Kokate, C. K. (1994). Practical Pharmacognosy. 4 [sup] th ed. New Delhi: Vallabh
Prakashan, 107.
20. Leckie, C. P., McAinsh, M. R., Allen, G. J., Sanders, D., & Hetherington, A. M. (1998).
Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose. Proceedings of
the National Academy of Sciences, 95(26), 15837-15842.
21. MacRobbie, E. A. C. (1981). Effects of ABA in ‘isolated’guard cells of Commelina
communis L. Journal of Experimental Botany, 32(3), 563-572.
22. MacRobbie, E. A. C. (1997). Signalling in guard cells and regulation of ion channel
activity. Journal of Experimental Botany, 515-528.
23. Manning, John; Goldblatt, Peter (2008). The Iris Family: Natural History &
Classification. Portland, Oregon: Timber Press. pp. 138–42.
24. McAinsh, M. R., & Hetherington, A. M. (1998). Encoding specificity in Ca2+ signalling
systems. Trends in Plant Science, 3(1), 32-36.
25. McAinsh, M. R., Brownlee, C., & Hetherington, A. M. (1990). Abscisic acid-induced
elevation of guard cell cytosolic Ca 2+ precedes stomatal closure. Nature, 343(6254),
186-188.
26. Mittelheuser, C. J., & Van Steveninck, R. F. M. (1969). Stomatal closure and inhibition
of transpiration induced by (RS)-abscisic acid. Nature, 221(5177), 281-282.
27. Ralph E. Brooks; Steven E. Clemants (2000). "Juncus". Magnoliophyta: Alismatidae,
Arecidae, Commelinidae (in part), and Zingiberidae. Flora of North America. 22. Oxford
University Press.
28. Scannell, M. J. (1973). Juncus planifolius R Br. in Ireland. The Irish Naturalists' Journal,
308-309..
29. Scannell, M. J., & Jebb, M. H. (2000). Flora of Connemara and the Burren: Records from
1984. Glasra, 4:7-45.
30. Watson, L. and M. J. Dallwitz. (2008). "Avena". The Grass Genera of the World.
Archived from the original on 2009-08-20. Retrieved 2009-08-19.
31. Zarinkamar, F., 2005. Foliar anatomy of Gladiolus segetum and Iris acutiloba (Iridaceae).
Proceedings of the 13th Iranian Biology Conference and the First International
Conference of Biology, Guilan University, Rasht, Iran.

32. Zarrinkamar, F., Jalili, A., Hamzehee, B., Asri, Y., Hodgson, J. G., Thompson, K., &
Shaw, S. (2002). FOLIAR ANATOMY OF CAREX IN ARASBARAN, NW.
IRAN. Iranian Journal of Botany, 9, 261-270.
33. Korn, R. W. (1972). Arrangement of stomata on the leaves of
Pelargonium zonale and Sedum stahlii. Annals of Botany, 36(2), 325-
333.
34. Essiett, U. A., &Iwok, E. S. (2014). Floral and leaf anatomy of Hibiscus
species. American Journal of medical and biological research, 2(5), 101-117.
35. Olotuah, O. F. (2014). Anatomy of leaf epidermis and petiole of two selected species of
Hibiscus. Res. J. Agric. Environ. Manage, 3(8), 403-405.
36. Osawaru, M. E., Dania-Ogbe, F. M., Chime, A. O., &Ogwu, M. C. (2011). Epidermal
morphology of west African okra Abelmoschus caillei (A. Chev.) Stevels from south
western Nigeria. Science World Journal, 6(3), 15-23.
37. https://www.gtac.edu.au/wp-content/uploads/2016/01/Leaf-Epidermal-
Peel_LabPreparation.pdf
38. Taratima, W., Ritmaha, T., Jongrungklang, N., Raso, S., & Maneerattanarungroj, P.
(2019). Leaf Anatomical Responses to Drought Stress Condition In Hybrid Sugarcane
Leaf (Saccharum Officinarum ‘Kk3’).
39. Santhakumari, C., & Krishnamurthy, K. V. (1991). Development of epidermis on banana
fruits. Biologia plantarum, 33(4), 325-331.
40. https://www.cbsetuts.com/ncert-class-10-science-lab-manual-stomata/
12.10- SUGGESTED READING

1. https://www.frontiersin.org/articles/10.3389/fpls.2021.716784/full#:~:text=The
%20stomatal%20index%20of%20the,of%20stomata%20and%20epidermal%20cells.
2. https://www.americangeosciences.org/word/stomatal-index
3. https://www.researchgate.net/publication/
284172830_Preparation_of_Epidermal_Peels_and_Guard_Cell_Protoplasts_for_Cellular
_Electrophysiological_and_-Omics_Assays_of_Guard_Cell_Function

4. https://www.researchgate.net/publication/
284172830_Preparation_of_Epidermal_Peels_and_Guard_Cell_Protoplasts_for_Cellular
_Electrophysiological_and_-Omics_Assays_of_Guard_Cell_Function
12.11- TERMINAL QUSETIONS

1. Define stomatal index by citing wheat leaf an example.
2. Describe the epidermal peel of Musain detail.
3. Give the details about the ABA exposure to the guard cells.
4. With the help of suitable diagrams, explain the epidermal peel of Tradescantia.
5. Describe different types of stomata found in angiosperms.

BLOCK-4 PLANT REPRODUCTION

UNIT-13-EXAMINATION OF MODES OF ANTHER

DEHISCENCE AND COLLECTION OF POLLEN
GRAINS FOR MICROSCOPIC EXAMINATION
13.1-Objectives
13.2-Introduction
13.3-Examination of modes of anther dehiscence and collection of pollen grains for microscopic
examination
13.4-Summary
13.5-Glossary
13.6-Self Assessment Question
13.7-References

13.1-OBJECTIVES
 Understand the male reproductive organ anther.
 Understand the modes of anther dehiscence.
 Understand the different types of anther attachment and anther dehiscence..
 Understand the techniques of pollen collection.
 Understand the techniques of pollen storage.
13.2 INTRODUCTION
An anther is a storehouse and manufacturing device of pollen, which plays structural and
functional key role in the reproduction of angiosperms. It is found at the top of the androecia or
more appropriately on the filament of the stamen. It produces the microspores or pollen grains,
stores them, and nourishes them until the time of dispersal. Thus, the anther is a part of the male
reproductive system or stamen inside the flower. Collectively, the stamens of a flower are termed
as androecium, and the number androecium can vary greatly between species to species; on the
basis of their numbers, plants are also classified as monandria, diandria triandria, tetrandria etc.
and can be classified on the basis of their arrangement as monadelphia, diadelphia, polydelphia,
tetradynamia, didynamia etc. There is also a high level of diversity associated with the position
and appearance of the stamens, they also may be fused at either the filament or the anther. The
male reproductive part or the structure of a flower typically consists of two elements; the slender
long filament and elliptical or cylindrical distal part anther. Generally, anther is a bilobed
structure which stores pollen grains in the pollen sacs (Fig. 13.1). The lobes of anther are
attached together by the connective. On the basis of modes of attachment to filament, anthers are
classified as, basifixed, adnate, dorsifixed and versatile (Fig. 13.2). The anther is a structure that
constitutes male reproductive part or structure of the flower known as “stamen” or “androecium”
by botanists (Fig.13.1). It has a distal knob-like structure, which generally consists of two lobes
joined together by the connective tissue known as “connective” in palynogy. After anthesis,
pollen grains get dispersed in the environment through dehiscence of anther. The pollen released
from anther reaches the stigma of either the same flower (self pollination) or of different flower
(cross pollination) during the process of pollination.

Fig.13.2: Modes of Anther attachment with filament.
13.3-EXAMINATION OF MODES OF ANTHER DEHISCENCE

AND COLLECTION OF POLLEN GRAINS FOR MICROSCOPIC
EXAMINATION
The splitting of anther at its maturity is known as anther dehiscence. Anther dehisces along the
line of delicacy to release its contents, which is referred to as pollens or microspores (Fig.13.3).
Anther dehiscence occurs either through detachment of a part of the anther or through special
opening pore (loculicidal). The dehiscence modes may be porous, short slits, longitudinal slits,
transverse, valvular and irregular (Fig.13.4). According Keijzert (1987), the pollen dispersal
requires fine tuning and regulation of development of the anther in a synchronized manner to
ensure that pollen release occurs at the precise time to maximize either cross or self-fertilization.
Anther dehiscence is the ultimate process and final function of the anther, which causes the
dispersal of pollen grains or microspores from pollen sacs. This process has finely tuned with
pollen differentiation, floral development, maturation and anthesis. The walls of anther splits at a

specific site called stomium, which is developed previously for dehiscence. These sites can be
seen as an indentation between the locules of each theca and runs the along the anther. In case of
plant species which have poricidal anther dehiscence, it is instead a fine pore. Anthers can be
classified as extrose and introse based on their facial arrangement. If it faces towards the
periphery of the flower it is called extrose and when the pollen releases from the anther by
splitting on the outer side, it is called extrorse dehiscence. If the face of anther lies towards
stigma or axis of the flower, then it is known as introse, and the pollens dispersed from the inner
side, is called introse dehiscence. If the pollen is released through a split that is positioned to the
side, towards other anthers, rather than towards the inside or outside of the flower, this
is latrorse dehiscence (Fig. 13.5).
Fig.13.3: Anther Dehiscence and release of Pollen Grains

Fig. 13.4: Different Modes of Anther Dehiscence
Fig.13.5: T. S. of Mature Dithecous Anther
COLLECTION OF POLLEN GRAINS

The primary requirement and crucial phase of pollen collection is to collect them in viable
condition. Viability is must be determined for any experimental study on pollen. Pollens are
collected soon after anther dehiscence for experimental studies to get optimal responses. In most
of plant species, it is convenient to detach flowers or whole inflorescences on the previous
evening and the cut end is dipped in water overnight in the laboratory. On the next day, the
anthers generally would have dehisced. Gently tap flowers on a watch glass and shed the pollen
grains. Another convenient method of pollen collection is to cut the mature anthers before
dehiscence and dehisce them under low humidity in desiccators and remove the remaining and
unnecessary part or debris with the help of pair of sterilized forceps or a brush. To prevent
contamination, it is advisable to choose mature undehisced anthers for collection and surface-
sterilize them with dilute chlorine water, or 10% sodium hypochlorite (NaOCl), or 0.25%

mercuric chloride (HgCl2), or 70% ethyl alcohol for 2 to5 min, then dry and lay them in a
sterilized petri dish for dehiscence.
Pollen storage:
Generally, pollen grains stored in glass or polythene vials (fig. 13.6) under low temperature and
suitable RH (relative humidity). For pollens stored in unsealed containers or vials, suitable
dehydrating agents like silica gel, sulphuric acid or solution of appropriate salts are used for
maintaining relative humidity. Lycopodium powder, wheat or corn flour and talcum powder are
used for preventing pollen grains from sticking together.
Non-polar organic solvents like benzene, diethyl ether and cyclohexane showed very less
leaching of phospholipids, sugars, and amino acids into the solvent and found suitable for
retention of viability. According to Jain and Shivanna (1988), polar organic solvents cause
extensive leaching of substance and viability loss of pollen grains. For long term storage, pollens
are freeze dried which involves the rapid freezing of pollen to sub-zero temperature of -60° C or
-80° C by use of inert gas like helium or nitrogen, followed by gradual removable of water under
vacuum sublimation. Long term preservation can also be done at ultra low temperature
(Cryopreservation) by use of dry ice (solid carbon dioxide), vapour phase nitrogen and liquid
nitrogen, temperature ranging between -70° C and -196° C like in pollen bank ( Fig. 13.7).
Fig.13.6: Different types of vials used in pollen collection and storage.

Fig.13.7: Liquid nitrogen canes used in cryopreservation.
13.4 SUMMARY
1. The anther is a store house and manufacturing device of pollen grains which plays
structural and functional key role in the reproduction of Angiosperms.
2. It produces the microspores or pollen grains, stores them, and nourishes them until the
time of dispersal.
3. The anther is a part of the male reproductive system or stamen inside the flower.
4. Collectively, the stamens of a flower are termed the androecium, and the number
androecium can vary greatly between species to species; on the basis of their numbers
plants.
5. There is also a high level of diversity associated with the position and appearance of the
stamens, they also may be fused at either the filament or the anther.
6. Generally Anther is a bilobed structure which stores pollen grains in the pollen sacs.
7. The lobes of anther attached together by connective, on the modes of attachment to
filament anther are classified as, basifixed, adnate, dorsifixed and versatile.
8. The anther is a structure constitutes male reproductive part or structure of the flower
known to as “Stamen” or “Androecium” by botanists.
9. It has a distal knob-like structure, generally which consists of two lobes joined together
by the connective tissue called connective in palynogy.
10. The pollen released from anther reaches on the stigma of either the same flower (Self
pollination) or in different flower (Cross pollination) during the process of pollination.

11. The splitting of anther at its maturity is called anther dehiscence, anther dehisces along a
built in line of delicacy in order to release its contents, called pollen or microspores.
12. Sometimes this involves the detachment of upper or some other parts of anther and
sometimes by special opening pore (Loculicidal).
13. The dehiscence modes may be porous, sort slits, longitudinal slits, transverse, valvular
and irregular.
14. Anther dehiscence is ultimate process and the final function of the anther which causes
the dispersal of pollen grains or microspores from pollen sacs.
15. The walls of anther splits at a specific site which are previously developed for dehiscence
called stomium.
16. Anthers can be classified as extrose and introse on their facial arrangement.
17. The primary requirement and crucial phase of pollen collection is to get them in viable
condition.
18. Generally, pollen collected soon after anther dehiscence for experimental studies to get
optimal responses.
19. On the next day the anthers generally would have dehisced, gently tap flowers on a watch
glass and sheds the pollen grains.
20. To prevent contamination it should be practiced to choose mature undehisced anthers for
collection and surface-sterilize them with dilute chlorine water, or 10% sodium
hypochlorite (NaOCl), or 0.25% mercuric chloride (HgCl2), or 70% ethyl alcohol for 2
to5 min, then dry and lay them in a sterilized Petri dish for dehiscence.
21. Generally Pollen grains stored in glass or polythene vials under low temperature and
suitable RH (relative humidity).
22. If pollens stored in unsealed containers or vials suitable dehydrating agents like silica gel,
sulphuric acid or solution of appropriate salts are used for maintaining relative humidity.
23. Lycopodium powder, wheat or corn flour and talcum powder are used for preventing of
pollen grains from sticking together.
24. Non-polar organic solvents like benzene, diethyl ether and cyclohexane showed very
little leaching of phospholipids, sugars, and amino acids into the solvent and found
suitable for retention of viability.

25. For long term storage pollen are freeze dried which; involves the rapid freezing of pollen
to sub-zero temperature of -60° C or -80° C by use of inert gas like helium or nitrogen.
26. Long term preservation can also be done by ultra low temperature (Cryopreservation) by
use of dry ice (solid carbon dioxide), vapour phase nitrogen and liquid nitrogen,
temperature ranging between -70° C and -196° C like in pollen bank.
13.5 GLOSSARY
Androecium: Collective term for stamen the male reproductive organ of plants
Anther: Top fertile portion of androecium that bears pollen sacs.
Anthesis: Opening of a flower bud.
Connective: The part of tissue that connects the anther lobes.
Cryopreservation: Preservation of cells or tissue at ultra low temperature.
Dehiscence: Splitting of anther at maturity.
Extrorse: Facing outward.
Introse: Facing inward.
Leaching: Removal or washing out of chemicals under the influence of solvent.
Palynology: Science deals with the study of pollen grains.
Pollen grains: Microspore or powdery mass of male reproductive cells.
Pollination: The process of transfer of pollen from anther to stigma.
Relative humidity: The ratio of the water vapor actually present in the air to the greatest amount
possible at the same temperature.
Stamen: The male reproductive part of a flower, usually with a slender filament supporting
the anther.
Stigma: The top flat part of pistil, modified for receiving pollens.
Stomium: The thin-walled cells of anther in the region of dehiscence.
Viability: Ability or capability of germination.
Vial: A small container, typically cylindrical and made of glass, or plastic used especially for
collection and storage of chemicals and cells.


1. Pollens are:
(a) Microspore cells (b) haploid cells
(c) Male cells (d) All of above
2. Pollens are produced in:
(a)Stigma (b) Anther
3. Pollen can be stored in liquid nitrogen for long term using the method of:
(a) Preservation (b) Cryopreservation
(c) Cryo-conservation (d) None of above
4. The ability of pollens to germinate is termed as:
(a) Non-viable (b) sterile
(c) Abortive (d) Viable
5. The splitting of anther at maturity is called:
(a) Cleavage (b) Splinter
(c) Fracture (d) dehiscence
6. Pollen test conducted in a test tube is called:
(a) Viability (b) fertility
(c) Sterility (d) All
7. Pollens facing inward or toward stigma are called:
(a) Introse (b) Extrose
(c) Latrose (d) None
8. Mercuric chloride (HgCl2) is used in:
(a) Viability test (b) Surface sterilization
(c) Pollen culture (d) All
9. The use of organic solvents for pollen storage leads to:
(a) Leaching (b) Chelation
(c) Aggregation (d) All
10. Lycopodium powder in pollen storage is used for:
(a) Preservative (b) Prevention of aggregation
(c) Sterilizer (d) Anti leaching agent

(1) Pollens facing outward of stigma called _______________.

(2) Pollens are produced inside of _________________.
(3) After maturation anther undergoes in the process of__________.
(4) Generally anther consists of two lobes which are joined together ___________.
(5) Dehiscence of anther occurs from specific region of anther called_______________.
(6) Removal or wash out of chemicals under influence of solvent ___________.
(7) Sulphuric acid or solution of appropriate salts is used for maintaining _______________in
pollen storage.
(8) Germination ability of pollen is called__________________.
(9) Anthers can be classified as extrose and ____________on their facial arrangement.
(10 Pollen sacs are found inside_____________.
(1) For maintaining relative humidity in stored pollen, sulphuric acid or solution of salts is used.
(2) Germination ability of pollen is called pollen sterility.
(3) Anthers are called extrose facial arrangement is outward.
(4) Germination ability of pollen is also called viablity.
(5) Pollens are collected from anther after dehiscens.
(6) Pollens are sterilized by boiling them in water.
(7) Polar organic solvents cause extensive leaching of substance and viability loss of pollen
grains.
(8) Lycopodium powder, wheat or corn flour and talcum powder are used for preventing of
pollen grains from sticking together.
(9) The walls of anther splits at a specific site which are previously developed for dehiscence
called stomium.
(10) The temperature of liquid nitrogen is -960C.

(1) Define microspore.
(2) What is dehiscence?
(3) Define extrose arrangement.
(4) Define viablity.

(5) What is called cryopreservation?

(6) What causes leaching of anther walls?
(7) Define stamen?
(8) Define anther.
(9) What is stomium.
(10) Define androecium.
13.6.1 Answer keys: 1-d, 2-b, 3-b, 4-d, 5-b, 6-a, 7-a, 8-a, 9-a, 10-b.
13.6.2 Answer key:1-extrose, 2-anther, 3-dehiscence, 4-connective, 5-stomium, 6- leaching,
7- relative humidity 8-viablity, 9- introse, 10- anther.
13.6.3 Answer key: 1-True, 2-False, 3-True, 4-True, 5-True, 6-False, 7-True, 8-True, 9-True,
10-False.
13.7 REFERENCES
 Amma, M. S. P. and Kulkarni A. R. 1979. Pollen storage in organic solvents. J. Palynol.
15: 100–104.
 Iwanami Y., Nakamura N. 1972. Storage in an organic solvent as a means for preserving
viability of pollen grains. Stain Technol. Vol.47(3):137-9.
 Jain A. and Shivanna K.R. 1988. Storage of Pollen Grains in Organic Solvents: Effect of
Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability.
Annals of Botany, Vol. 61(3): 325–330.
 Keijzert C. J. 1987. The processes of anther dehiscence and pollen dispersal. New
Phytologist. Vol. 105(3): 499-507.

 Geitmann A. (Edt). 2020. Pollen and Pollen Tube Biology: Methods and Protocols
(Methods in Molecular Biology) 2020.
 Shivanna, K.R. and Rangaswamy, N.S.1992. Pollen Biology: A Laboratory Manual.
Springer.
 Stanley R. G. and Linskens H. F. 1974. Pollen: Biology Biochemistry Management.
Springer.

(1) Write short note on anther.
(2) Describe microspore in brief.

(3) What do you understand by androecium?
(4) Describe cryopreservation.
(5) Differentiate between an anther and a filament.
(6) Describe the methods used for storage of pollens.
(7) Write a short note on anther attachment.
(8) What do you understand by stomium?
(9) Write a note on introse and extrose arrangement of anther.
(10) Define pollen storage techniques.

(1) Write a note on stamen and its part.
(2) Describe different modes of anther dehiscence with suitable diagram.
(3) Write a detailed note on pollen collection and storage.
(4) Write a note on the use of polar and non polar solvent and their drawbacks in pollen storage.
(5) Describe the methods used in pollen storage.

UNIT-14-TESTS FOR POLLEN VIABILITY USING STAINS

AND IN-VITRO GERMINATION, USING HANGING DROP AND
SITTING DROP CULTURES
14.1-Objectives
14.2-Introduction
14.3-Tests for pollen viability using stains and in-vitro germination
14.4-Pollen germination using hanging drop and sitting drop cultures
14.5-Summary
14.6-Glossary
14.8-References

14.1-OBJECTIVES
 Understand the concept of pollen physiology.

 Understand the pollen viability.
 Understand the methods of pollen germination.
 Understand in vitro and in-vivo pollen germination test.
14.2 INTRODUCTION
The term ‘pollen’ is usually used to describe male gametes of spermatophytes or powdery mass
of microspores. Pollens are the male reproductive cells, which represents the male gametophytic
generation of spermatophytes (seed-bearing plants) that produce male gametes (sperm cells) on
germination. In gymnosperms, pollen grains or microspores are produced within
microsporangium of male cones. In angiosperms, pollen grains are produced inside anthers. The
process of pollen formation is called microsporogenesis.
The plant body of angiosperms is diploid and specialized organs for reproduction are present in
flower. Stamens are the male reproductive organs. In most angiosperms, each stamen is
composed of an anther that has anther lobes and a filament. The anther mostly contains four
microsporangia which are joined by the connective. The anther wall consists of four layers: (i)
the epidermis (exothecium) (ii) endothecium (iii) middle layer(s) and (iv) tapetum. In each
microsporangium, the central region contains diploid microspore mother cells or pollen mother
cell, which eventually forms the pollen grains. Each microspore mother cell undergoes meiosis
and result in the formation of four haploid cells called tetrads. The microspores start to
differentiate while associated in tetrads and give rise to four pollen grains. The second phase is
micro gametogenesis, wherein pollen grain undergoes mitotic division and forms a large
vegetative cell and a small generative cell. The generative cell in most of the species divides to
form two sperm cells before the germination of pollen tube. Pollen grains are shed from anther
and transferred or carried to the stigma by various agencies like wind, insect, water etc. After
reaching on stigma, pollen germinates and produces pollen tube through the germ pore. Pollen
tube grows from its tip and travels through style and reaches the embryo sac and finally enters
the ovule, where it enters into embryo sac. The male nuclei of pollen tube fuse with egg and
polar nuclei, which is called fertilization. Pollen germination on stigma is a complex process,

where it is identified genetically and the whole process is species specific. All pollens cannot
germinate and those pollens that are able to germinate on stigma are called viable pollens. The
germination ability of pollens can be tested chemically in laboratory.
13.3- TESTS FOR POLLEN VIABILITY USING STAINS AND IN-

VITRO GERMINATION
Pollen Viability
Pollen viability of is described as the capability of germination and is a measure of male fertility.
Viable pollen is responsible for a high fertility rate and crop yield. In crop hybridization
programmes, pollen fertility and viability have a paramount importance. Pollen viability also
plays a key role in germplasm or pollen bank. Pollen can be stored for germplasm conservation,
conduction of hybridization experiment between plants that flower at different times or places,
and for later use in hybridization programmes. For such experiment, the quality of pollens must
be maintained according to Kearns and Inouye (1993).
Pollen Viability Tests:
The pollen viability can be determined by direct and indirect methods. In the direct (in vivo)
method, pollens are transferred on receptive stigmas and to determine whether seeds are
produced or not. Direct testing methods has the advantage of providing an unequivocal measure
for the population of pollen grains deposited on the stigma, but it has several disadvantages as it
is tedious, time-consuming, labour intensive and requires lager number of fresh samples. Pollen
viability or germination percentage can also be calculated by in vitro techniques. Indirect
methods rely on the correlation between ability to fertilize an ovule and some physiological or
physical characteristics that can be determined faster. Indirect spacing methods that correlate
with pollen germination include (1) the fluorochromatic procedure (FCR), (2) testing pollen for
enzyme activity, and (3) testing stain ability of vegetative cells.
Pollen Staining Tests
Stains are chemical dyes that are used for particular type of cells and cell organelles. Some stains
are specific to pollen components and can be used as a viability indicator or for testing pollen
viability. According to Dafni et al. (2005), nuclear satin acetocarmine stains chromosomes;
aniline blue with lactophenol stains callose, phloxin-methyl green stains cellulose and cytoplasm

of cell. However, Heslop-Harrison (1985) demonstrated that immature or non-viable pollen

sometimes contains enough of these elements that also shows staining properties and viable
pollen of some species do not stain well. Therefore, stains provide (at best) only rough estimates
of viability.
Some stains such as acetocarmine in glycerol jelly and aniline blue in lactophenol stain viable
pollens but don’t stain non-viable or abortive pollens. However, an improved Alexander's stain
can be used as differential stain for pollens. Alexander’s stain uses chloral hydrate, phenol and
mercuric chloride, all of which are highly toxic. Peterson et. al., (2010) modified Alexander's
stain (1969) technique and improved pollen staining technique by not using a regulated chemical
chloral hydrate, mercuric chloride and phenol. This technique requires a much shorter time
period for sample preparation and staining. This simplified method is very useful for field studies
of angiosperm as well as gymnosperm pollens without high-end equipments such as fluorescence
microscopes. It differentially colors abortive and non-abortive pollen; malachite green stains
cellulose in pollen walls, and acid fuchsin stains protoplasm. Thus abortive (or germinated)
grains appear green, and grains with protoplasm appear pink. Alexander's (1969) stain is also
used to distinguish self versus outcross pollen on stigmas in species, where the incompatibility
mechanism acts at the stages of pollen germination and self-sterile pollen retains cytoplasm.
Following are some staining tests and protocols for pollen grains viability assessment.
Tetrazolium Test: The Tetrazolium (2,3,5 triphenyl tetrazolium chloride) test is commonly
known as the TTC Test or TZ test. Norton, (1966) demonstrated that all living cells, which
respire, are capable of reducing a colourless chemical TTC (2, 3, 5 triphenyl tetrazolium
chloride) into a red-colored compound formation by H+ ion transfer reactions catalysed by
enzyme dehydrogenases. According to Shivanna and Johri (1989), the test has provided accurate
results for many taxa. However Sedgley and Harbard, (1993) proved this method has some
drawbacks like it tends to overestimate viability because sometimes it also stains non-viable
pollen grains. The concentration of tetrazolium salt, temperature and period of incubation needs
to be standardized to get optimal results in various pollen samples.
Procedure: 1% TTC was prepared by adding 0.2 g. TTC and 12 g sucrose dissolved in 20 ml dd
H2O. Drop two drops of the mixture on a slide and transfer pollen over it and place a cover glass.

Count colored viable pollens after 30-40 minutes of incubation at 400 C in a dark place. Pollen
grains that stained orange or bright -red color were counted as viable.
Iodine-potassium iodide Test (I2KI): This technique indicates viability and determines the
starch content of pollen-grains. On staining with this, Iodine in a watery arrangement of
potassium iodide breaks up the tri-iodide-anion edifices with starch and giving viable indicator
of blue-black color to pollen grains.
Procedure: Dissolve 1 g potassium iodide and 0.5 g iodine in dd H2O and finally prepare volume
of 100 ml. Drop 1 or 2 drops of the dye over pollen sample and mix evenly in a gentle
manner. Place a cover glass over it and after 5-10 minutes count the number of stained (viable)
pollen grains under the microscope.
Aceto-carmine test (2%): Aceto-carmine is a nuclear stain that is used for staining nucleated
live cells. The pollen nucleus is rich in chromatin material and viable pollen stains pink to deep
red with aceto-carmine, whereas non-viable pollen grains do not take any stain.
Procedure: Weigh 2 g of carmine powder, dissolve it in 95 ml of glacial acetic acid. Add dd

H2O final 100 ml volume solution. Boil it and let it cool down. Filter it with the help of a
sterilized filter paper and store it in a cool and dark place. Take two to three drops of stain on
slide and transfer pollen grains on it and place a coverslip on it. Count stained viable pollens
after 5-10 min.
Aniline blue (Cotton blue) test: Hauser and Morrison (1964) demonstrated that aniline blue
stain detects the callose of the pollen walls and pollen tubes. The solution is prepared by adding
200mg/lit of aniline blue in a mixture of 10ml each of phenol, lactic acid, glycerol and distilled
water. The viable pollen stained dark blue color, while dead pollen are unstained.
Modified Alexander method: Peterson et al., (2010) modified Alexander’s stain (1969) which
clearly differentiates between aborted and non-aborted pollens. Acid fuchsin, present in this dye,
stains the protoplasm and malachite green stains cellulose in walls of pollen; dark purple pollen
was scored as viable and green as aborted. The non-aborted pollen grains stained magenta-red
and aborted pollen grains stained blue-green.
Procedure: This stain is prepared by adding 10ml of 95% ethanol, 1 ml of Malachite green (1%
solution in 95% alcohol), 50 ml dd H2O, 25 ml glycerol, 5 ml acid fuchsin (1% solution in

water), 0.5 ml orange G (1% solution in water), 4 ml glacial acetic acid and add dd H2O (4.5 ml)
to final volume of 100 ml. Flower buds are fixed in 2 hour in Carnoy’s fixative (6 alcohol:3
chloroform:1 acetic acid) before anthesis. The flower buds or pollen grains are dried and mixed
with 2-4 drops of stain. Once the sample is stained, slowly heat the slide over flame until the
stain solution starts boiling (~30 seconds). A more moderate rate of heating allows better
penetration of the dye into the cellulose and protoplasm of the pollen. Place a cover slip over it
and observe it under the microscope. The per cent pollen viability was calculated using formula:
In-Vitro Pollen Germination:
In-vitro pollen germination technique is commonly used in physiology it provides a simple clear
experimental method to study the physiology and biochemistry of pollen germination and growth
of pollen tube, as well as the responses of the pollen system to physical and chemical factors. In-
vitro techniques are generally accomplished within a few hours from pollen culture except in
some cases. Maintenance of sterilized environment and aseptic conditions for routine exercises is
not a necessity. But in case of pollens that take longer period of germination, such pollen cultures
have to be free from microbial contamination, and therefore aseptic culture techniques have to be
used. The culture medium can be sterilized by autoclave or sterile Millipore filter unit (pore size
0.2 μm). Antibacterial and antifungal substances such as rifampicin (antifungal) and nistatin
(antibacterial) (10–15 μg/ml) can be incorporated in the germination medium. Before culturing
the pollen grains, they are left in humid environment; high humidity improves the germination
ability of pollen grains. This is achieved by dusting of pollen grains uniformly on a microslide,
then incubating them for 15-60 minutes on a moist filter paper (>90% RH). If it is difficult to
spread the pollen (in case of sticky pollen grains especially of entomophilous species), treat them
with an organic solvent, such as cyclohexane or hexane for 2-3 minutes, then air-drying them for
5-l0 min prior to culturing. Culture medium plays a key role in pollen germination; composition
of a germination medium to obtain optimal responses has to be empirically formulated for each
species. Generally three constituents viz. sucrose, boric acid, and calcium nitrate, are sufficient
for pollen germination and growth. However, the optimal concentration of sucrose varies on the
type of species, 100 mg/I boric acid and 300 mg/I calcium nitrate are optimal for pollens of most

species. Brewbaker and Kwack's Medium, Roberts' Medium and Hodgkin and Lyon's Media are
suitable and support most of pollen grains for culture and growth. Composition of different
media is given as below:
Brewbaker and Kwack's Medium (Brewbaker and Kwack 1963):

Sucrose 10%
Boric acid: 100mg/l
Calcium nitrate: 300mg/l
Magnesium sulfate: 200mg/l
Potassium nitrate: 100mg/l
Roberts' Medium (Roberts et al. 1983):
Sucrose: 20%
Boric acid: l0mg/l
Calcium chloride: 362mg/l
Potassium nitrate: 100 mg/l
Tris: 60-130mg/1
Hodgkin and Lyon's Medium (Hodgkin and Lyon 1986):
Sucrose 580 mM (ca. 20%)
Boric acid 1.62 mM (ca. 100 mg/I)
Calcium nitrate 1.69 mM (ca. 400 mg/I)
Potassium nitrate 0.99mM (ca. 100mg/l)
Magnesium sulfate 0.84mM (ca. 200mg/l)
TAPS 20mM (ca. 4.86g/l)
14.4 POLLEN GERMINATION USING HANGING DROP AND

SITTING DROP CULTURES
Germination Test
Pollen grains of most species germinate and grow in a tube when they are placed in appropriate
solution of calcium, boron, and sucrose. Although it provides a controlled experimental system,
germination in-vitro does not completely mimic growth in in-vivo. Some factors affect them such
as temperature and germination media. These can also be affected by time of collection and
storage conditions as well as culture media and pollen density on the media. A large numbers of

pollens have been successfully germinated by palynologists/physiologists under controlled

laboratory conditions on suitable media. Pollen germination in the stigma (in-vivo) was first
observed as early as 1824 by C. B. Amici in Portulaca, who later observed the germinating
pollen tube entering ovule.
There are several methods of in-vitro raised by palynologists/physiologists for pollen cultures
according to requirement of particular species. The selection of a procedure depends on
requirement of the particular species and also the nature of study and the facility available. The
two mostly used methods are as follows:
Hanging Drop Culture
The hanging drop culture method is an old fashioned. In the hanging drop method of pollen
germination, the growth of the pollen tube was determined by using a drop of germinating media
on a cover glass; pollen grains are dusted on the media drops with the help of a sterilized and
clean brush, and then the cover glass is inertly placed on the cavity slide. It is then left in an
incubator under dark conditions at 25°C temperature in a culture medium with approximate
composition of 5% sucrose, 5 ppm boric acid (H3BO3), and 1% agar for 1 day, 2 days and 3
days (24, 48, and 72 hours of time). For each incubation period, observe and record germination
in three drops by counting three fields. A pollen grain will considered, if the length of
germinated pollen tube was at least equal to or greater than the grain diameter. Measurements of
pollen tube length are recorded directly with the help of micrometry slide or eyepiece of the
microscope. The mean length of the pollen tube is calculated from each drop.

Fig.1: Hanging drop culture plate with several drop cultures
Fig.2: Hanging drop culture plate with slide
Sitting Drop Culture
The sitting drop culture method of pollen grains is a simpler one than the hanging drop method
of pollen culture. The sitting drop culture method of pollen grains involves culturing of pollen
grains in a drop of culture medium which is placed on a microslide. The culture is then
transferred to in a humid chamber under controlled temperature to prevent evaporation. The
constitution of the culture medium is the same as used in the hanging drop method.
14.5 SUMMARY
1. The term ‘pollen’ is usually used to describe male gametes of spermatophytes or powdery
mass of pollen grains or microspores.
2. Pollen contains the male reproductive cells, which represents the male gametophytic
generation of spermatophytes.
3. The process of pollen/microspore formation is called microsporogenesis.
4. The stamens are the male reproductive organs.
5. The anther mostly contains four microsporangia joined by the connective.
6. In each microsporangium, the central region contains a diploid microspore mother cell or
pollen mother cell, which eventually forms the pollen grains.
7. Microspore mother cell undergoes meiosis that results in the formation of four haploid cells
or tetrads.
8. Pollen grains are shed from anther and transferred or carried to the stigma by various
agencies like wind, insect, water etc.

9. After reaching on the stigma, pollen germinates and produces the pollen tube through the
germ pore.
10. Pollen tube grows from its tip and travels through style and reaches the embryo sac.
11. All pollens cannot germinate and the pollens that are able to germinate on stigma are called
viable pollens.
12. Pollen viability is described as the capability of germination and is one of the measures to
determine male fertility.
13. The germination ability of pollens can be tested chemically in laboratory.
14. Pollen viability also plays a key role in germplasm or pollen bank; pollen can be stored for
germplasm conservation and for the conduction of hybridization experiment.
15. The pollen viability can be determined by direct and indirect methods.
16. Direct testing method has the advantage of providing an unequivocal measure for the
population of pollen grains deposited on the stigma, but it has several disadvantages as it is
tedious, time consuming and labor-intensive and requires lager number of fresh samples.
17. Pollen viability or germination percentage can also be calculated by in-vitro techniques.
18. Stains are chemical dyes used for a particular type of cells and cell organelles.
19. Some stains such as acetocarmine in glycerol jelly and aniline blue in lactophenol stain
viable pollens but don’t stain non-viable or abortive pollens.
20. In-vitro pollen germination technique commonly used in physiology.
21. In-vitro techniques are generally accomplished in short time period i.e. within a few hours
from pollen culture except in some cases.
22. Pollen germination in the stigma (in-vivo) was first observed as early as 1824 by C. B. Amici
in Portulaca who later observed the germinating pollen tube entering the ovule.
23. There are several methods of in-vitro raised by palynologists for pollen cultures according to
requirement of particular species.
24. The selection of a procedure depends on requirement of species and the nature of the study
and facility available. The two most commonly used methods are Hanging drop culture and
sitting drop culture method.

14.6 GLOSSARY
Anther: The part of flower that produces and contains flower.
Culture media: Any specific mixture of nutrients and other substances that supports growth.
Gametophytic: Gamete producing or haploid plant.
In-vitro: In glasswares or test tube.
In-vivo: In natural condition or within organism.
Incubator: A safe, controlled apparatus that provides necessary environment for growth and
development of organ and organism.
Microspore: Male gamete or pollen grain in case of an angiosperm.
Palynology: Science that deals with the study of pollen grains.
Pollen: Mass of microspores in spermatophytes that usually appears in form of a fine dust.
Pollen mother cell: A diploid cell within anther or pollen sac which gives haploid pollen by
meiotic division.
Pollen tube: A hollow tube that develops from a germinating pollen grain.
Protocol: A predefined procedure or method in implementation of an experiment.
Spermatophytes: Seed-producing plants.
Stain: A chemical dye used for coloration in a particular tissue or cells.
Stigma: Tip of pistil/pistils adapted for receiving pollen.
Viability: Ability to germinate or maintain itself.
14.7 SELF ASSESSMENT QUESTION

1. Pollen are:
(a) Microspore cells (b) haploid cells
(c) Male cells (d)All of above
2. Pollens are produced in:

(a)Stigma (b) Anther

3. Pollen tube grows from:
(a) Base (b) Tip
(c) Intercalary (d) None of above
4. Pollens that are able to germinate are called:
(a) Non-viable (b) sterile
(c) Abortive (d) Viable
5. Pollen on germination gives rise to:
(a) Root (b) Stem
(c) Cotyledons (d) Pollen tube
6. Pollen viability test in a test tube is called:
(a) In-vitro (b) In-vivo
(c) In-situ (d) All
7. Brewbaker and Kwack's Medium does not contain:
(a) TRIS (b) Sucrose
(c) Boric acid (d) Calcium Nitrate
8. In a viability test, acetocarmine stains:
(a) Chromosomes (b) Callose
(c) Pollen Wall (d) All
9. Tetrazolium (2,3,5 triphenyl tetrazolium chloride) is a:
(a) Stain (b) Nutrient
(c) Enzyme (d)All
10. Aniline blue stains:
(a) Callose (b) Chromosomes
(c) Cytoplasm (d) Nucleus

(1) In-vivo pollen germination was observed for the first time by _______________.
(2) Pollen germination experiment is carried out in glassware called _________________.
(3) After reaching stigma, pollen germinates and produces __________.

(4) ________________ grows from its tips and travels through style and reaches the embryo sac.
(5) In ________________ method of pollen germination, pollen tube growth was determined by
use of a drop of germinating media on a cover glass.
(6) Tris is an additional supplement of________________ media.
(7) TAPS is an additional supplement of________________ media.
(8) Germination ability of pollen is called__________________.
(9) Iodine-potassium iodide Test (I2KI) technique indicates _____________ content of pollen-
grains.
(10) Aceto-carmine is an_____________ stain that stains nucleated live cells.

(1) In-vivo pollen germination was observed for the first time by C. B. Amici in Portulaca.
(2) Aniline blue stains chromosomes.
(3) Experiment carried out in a test tube or a glassware called in-vitro.
(4) Germination ability of a pollen is also called viability.
(5) Pollen viability test can be determined with the help of staining tests.
(6) The meristem situated at the bases of internodes is known as apical meristem.
(7) In the hanging drop method of pollen germination, pollen tube growths were determined by
using the drop of germinating media on a cover glass.
(8) Aniline blue with lactophenol stains callose, phloxin-methyl green stains cellulose and
cytoplasm of cell.
(9) Aceto-carmine is a nuclear stain that stains nucleated live cells.
(10) Pollen tube grows from base of tube.

(1) Define microspore.
(2) What is palynology?

(3) What is tetrazolium?
(4) Define viability.
(5) Define culture media.
(6) What is the name of the nuclear stain?
(7) What is in-vitro?
(8) Define protocol.
(9) Describe pollen tube.
(10) Define in-vivo.
14.7.1 Answer keys: 1-d, 2-b, 3-b, 4-d, 5-b, 6-a, 7-a, 8-a, 9-a, 10-a.
14.7.2 Answer key: 1-C.B. Amici, 2-in-vitro, 3-Pollen tube, 4- Pollen tube, 5- Hanging drop,
6- Roberts' Medium, 7-Hodgkin and Lyon's Medium, 8-Viablity, 9- viability and
starch, 10- nuclear.
10-False.
14.8 REFERENCES
 Alexander MP. 1969. Differential staining of aborted and nonaborted pollen.
StainTechnol. 44:117-22.
 Brewbaker, J.L. and Kwack, B.H., 1963. The essential role of calcium ion in pollen
germination and pollen tube growth. American Journal of Botany, 50, 859–865.
 Dafni, A., Kevan, P.G., and Husband, B.C., 2005. Practical Pollination Biology. Environ
quest Ltd. Ontario.
 Hauser E.J.P. and Morrison J.H., 1964. The cytochemical reduction of nitroblue
tetrazolium as an index of pollen viability. Amer. Journ. Bot., 51: 748-752.
 Heslop-Harrison, J. and Heslop-Harrison, Y., 1985. Germination of stress-tolerant
Eucalyptus pollen. Journal of cell science, 73, 135–57.

 Hodgkin T, Lyon GD .198. The effect of Brassica oleracea stigma extracts on the
germination of B. oleracea pollen in a thin layer Chromatographie bioassay. J Exp Bot
37:406–411
 Kearns, C.A. and Inouye, D.W., 1993. Techniques for pollination biologists. Colorado,
USA.: University Press of Colorado, Niwot.
 Norton D J. Testing of plum pollen viability with tetrazolium salts. American Society for
Horticultural Science. 1966; 89:132–134.
 Peterson R, Slovin JP, Chen C. 2010. A simplified method for differential staining of
aborted and non-abortedpollen grains. International Journal of Plant Biology: 1(2): 66-69.
 Roberts AB, Lamb CC, Newton DL, Sporn MB, De Larco JE, Todaro GJ .1983.
Transforming growth factors from neoplastic and non-neoplastic tissues. Fed Proc 42:
2621–2626.
 Sedgely M. and Harbard J. 1993. Pollen storage and breeding systems in relation to
controlled pollination of four species of Acacia (Leguminosae: Mimosoideae) Australian
Journal of Botany, 41: 601-609.
 Shivanna, K.R. and Johri, B.M., 1989. The Angiosperm Pollen: Structure and Function.
Wiley Eastern Ltd., New Delhi.

 Shivanna K.R. and Rangaswamy N.S. 1992. Pollen Biology: A Laboratory Manual. Springer.
Pp119.
 Johri B.M. and Srivastava P.S. 2001. Reproductive Biology of Plants. Springer. Pp 320.
 Shivanna K.R. 2003. Pollen Biology and Biotechnology. CRC Press. Pp 316.
 Geitmann, A. 2020. Pollen and Pollen Tube Biology: Methods and Protocols. Pp 325.

(1) Write a short note on in-vitro pollen germination.
(2) Describe the hanging drop method of pollen culture.
(3) What do you understand by a stain?

(4) Describe a few pollen staining techniques.
(5) Differentiate between the hanging drop and the sitting drop method of pollen culture.
(6) Differentiate between in-vivo and in-vitro methods.
(7) Write a short note on pollen viability.
(8) What do you understand by culture media?
(9) Write a note on pollen tube.
(10) Describe Brewbaker and Kwack's Medium.
(1) Write a detailed note on Germination Test.
(2) Describe in-vitro methods of pollen germination with protocols.
(3) Write a detailed note on pollen staining viability test.
(4) Write a note about on modified Alexender’s test.
(5) Describe in-vitro pollen culture media.
UNIT-15-STUDY OF THE PRIMARY AND SECONDARY

ABNORMAL GROWTH IN PLANTS
15.1-Objectives
15.2-Introduction
15.3- Study of the primary and secondary abnormal growth in plants
15.4-Summary
15.5-Glossary
15.6-Self Assessment Question
15.7-References

15.1-OBJECTIVES
 Understand the phenomenon of plant growth.

 Understand the plant growth types.
 Understand the primary and secondary growth in plants.
 Understand the abnormal growth in plants.
 Understand the primary and secondary abnormal growth in plants.

15.2- INTRODUCTION
In plants increase in size and length of stems and roots is called as growth. The stems and roots
of plants, continue to grow throughout life span and this is called indeterminate growth. On other
hand, some plant parts, such as, spines, leaves and flowers, the growth ceased after their
development, such growth pattern called determinate growth. According to Cutler et. al., (2008)
the plant part or cells which are responsible for growth and repair are meristems and such cells
are called meristematic cells. Meristem is of undifferentiated mass of cells, capable of divide
continue. As plant cells divide and metamorphosed into special cells for particular function
through cellular differentiation. Apical meristems are found at the tip or apex and responsible for
growth in length. Apical meristem on division gives rise to new cells which allow growth in
length or height, which is known as primary growth.
According to Evers (1982) secondary meristems on division and differentiation allow growth in
diameter in woody dicots and gymnosperms. Monocots do not have cambium thus secondary
growth is not found in such plant. Cork cambium and vascular cambium (Fig.15.1) are two types
of secondary meristem produces which are responsible for growth to the diameter of the plant
stem called secondary growth.
Fig.15.1: Position of Vascular Cambium in Dicots (I) Fascicular Cambium (II) Interfascicular
Cambium

15.3- STUDY OF THE PRIMARY AND SECONDARY ABNORMAL

GROWTH IN PLANTS
Primary growth in plants occurs at the apices, apical meristem causes the increase of stem length.
Apical meristems differentiate into the three types of basic meristematic tissues the protoderm
(produces epidermal layers), ground meristem (produces ground tissue system), and
procambium (produces vascular tissue). The growth caused by these tissues is considered as
primary growth. Primary growth occurs when plants grow upward or toward the sunlight
necessary for photosynthesis and also downward or geotropically to sink roots deep into the
soil to anchor absorb water and nutrients. This 'up and down' growth is possible due to apical
meristem that, upon division, produces undifferentiated cell that will become either a new root
or shoot tip.
Secondary growth in plant occurs when stems or branches grow outward by activity of lateral
meristem. Unlike the primary meristem which causes the plant to grow up or down, lateral
meristematic tissue causes the plant to increase in girth by adding growth rings. Thus the
growth in width or circumference of trees or increases in girth is termed as secondary growth
and it arises from cambium. As with apical meristems, lateral meristems are regions of high
cell division activity. Dicots and Gymnosperms posses cambium thus secondary growth is
possible only dicots, monocots, however, do not experience secondary growth due to lack of
cambium, But exceptionally in some monocots such as Palm, Yucca, Dracaena, Smilax,
Agave, Coconut etc, secondary growth takes place.
Fig. 15.2: Position of Vascular cambium and Cork Cambium

Fig15.3: Cork cambium in Dicot Stem (A) Formation of first layer of cork cells, (B)
Cork formed in multiple layers by the activity of cork Cambium.
The two important factors for secondary growth are the vascular cambium and the cork
cambium (Fig.15.2&15.3). The vascular cambium produces more vascular tissue i.e. xylem and
phloem, which provides support for the shoot system in addition to transporting water and
nutrients. Generally cambium functions normally and plant grows too normally in dicots but
sometimes behavior of cambium changes and causes unequal growth pattern which is called
abnormal secondary growth. There may be various factors which causes abnormal growth in
plants.
Anomalous structures in Plants:
In Several dicots secondary growth deviates considerably from the normal secondary growth
such secondary thickenings are called abnormal or anomalous, these anomalies or abnormalities
may be as follows:
I. Following reasons are responsible for abnormal secondary growth in dicots:

(a) Anomalous position of vascular cambium
(b) Abnormal Activity of vascular cambium
(c) Formation of accessory vascular cambium
(d) Formation of extrastelar cambium
(e) Formation of interxylary phloem
II. Presence of scattered vascular bundles in dicots
III. Presence of exclusive phloem and xylem bundles

IV. Presence of medullary bundles

V. Presence of extra stellar or cortical bundles
VI. Ring arrangement of vascular bundles in monocots
I. (a) Anomalous/abnormal position of vascular cambium: Normally vascular

cambium is circular, but in stem of some plants it is folded. Later on these folds
break and separate from each other. Each fold is responsible to form a complete
vascular bundle. Many vascular bundles are formed in stem e.g. Serjania,
Thinouia and Bauhinia (fig.15.4).
Fig,15.4: Abnormal position of vascular cambium showing in T.S. of (A)Serjania, (B)

Thinouia and (C) Bauhinia Stem.
I. (b) Abnormal Activity of vascular cambium: Generally, xylem and phloem

tissues are formed from the maximum part of the vascular cambium and medullary
rays are formed from the few parts of vascular cambium but in some plants
parenchyma (medullary rays) are formed from the maximum part of the vascular
cambium and rarely in some places xylem and phloem tissues are formed e.g.
Aristolochia (Fig15.5).

Fig15.5: T. S. of Aristolochia stem showing abnormal activity of vascular cambium
I. (c) Sequential (successive) ring of vascular cambium: In some of the plants, a

new ring of vascular cambium is formed each year. According to Esau (1969) this is
formed outside the previous ring e.g. Mirabilis, Boerhaavia, Bougainvillea etc
(Fig15.6).
Fig.15.6: Sequential (successive) ring of vascular cambium in T.S. of Mirabilis,

Boerhaavia and Bougainvillea stem.
I. (d) Formation of vascular cambium from pericycle: Vascular cambium is formed

from the pericycle in plants of amaranthaceae and ahenopodiaceae families. A complete
ring of vascular cambium is formed from the pericycle.

I. (e) Interxylary phloem or included phloem: This is also called internal phloem or
included phloem. The origin of interxylary phloem (Fig15.7) in most plants is primary.
The internal phloem develops after the development of external primary phloem. This
type of development is found in plant families solanaceae, apocynaceae and
lathyraceae.
Fig.15.7: T. S. Calotropis (Apocynaceae) stem showing Included phloem.
II. Scattered vascular bundles in dicotyledons: In dicots vascular bundles found normally
arranged in a ring but in some plants such as Thalictrum, Piper, Peperomia, Podophyllum,
Papaver etc. vascular bundles are scattered (fig15.8).
Fig.15. 8: T.S of in Peperomia stem showing scattered vascular bundles.

III. Presence of exclusive phloem and xylem bundles: Sometimes vascular bundles are
incomplete i.e. a bundle is represented either exclusively by xylem or phloem strand. In
Paeonia, in addition to normal vascular bundle, incomplete bundles are also present

which are exclusively represented by xylem. Similarly, in Cuscuta, Boerhaavia,

Ricinus, and Antigonon only phloem bundles are present.
IV. Presence of medullary bundles: In some dicots e.g. members of Ranunculaceae,

Amaranthaceae, Acanthaceae, Cactaceae and Chenopodiaceae vascular bundles are
present in pith (Fig15.9) and then they are known as medullary bundles and show a
limited amount of secondary growth.
Fig.15.9:T.S.of Mirabilis Stem showing Medullary bundles.
V. Presence of cortical bundles: In some dicots in addition to the normal ring of stelar
bundles some vascular bundles are also present in the cortex known as cortical bundles
e.g. Casuarina, Nyctanthes (Fig15.10).

Fig.15.10: T. S. Nyctanthes Stem showing Cortical bundles.
VI. Ring arrangement of Vascular bundles in monocots: In monocots vascular bundles

scattered in the ground tissue, but in some cases like Dioscorea vascular bundles found
arranged in two rings around the pith anomously; the outer ring has only two small
bundles which are embedded in the sclerenchymatous pericycle and the inner ring has
several large vascular inside the pericycle.
Types of anomalous secondary growth:
The anamalous secondary growth can be divided in two types:
1. Abnormal growth from abnormal cambium in monocots
2. Abnormal growth from normal cambium in dicots
1) Abnormal growth from abnormal cambium ( e.g. Secondary Thickening in Dracaena
stem: Monocot):
The secondary thickenings normally absent in monocot plants since the vascular bundles in
monocots are without cambium (closed type). However, a very few plants in monocots shows
anomalous secondary growth such as Dracaena, Yucca, Aloe, Sansevieria and Agave. Here you
will study this by example of anomalous secondary growth in Dracaena stem.

In monocotyledons normally the vascular bundles are closed. The cambium being absent the
secondary growth is absent; but but later on a secondry meristem develops which behaves as
cambium like Dracaena and Yucca secondary growth takes place. This abnormal cambium may
either develop from cortex or pericycle and shows abnormal activity.
The young stem has typical structure i.e. epidermis is followed by sclerenchymatous hypodermis
and large number of closely arranged vascular bundles scattered in ground tissue. The outer layer
of cells from the ground tissue becomes meristematic and functions as cambium. The cambium
formed in the region which has ceased elongation. The activity of this cambium is more on the
inner side and very little on the outside where it forms only parenchyma. On the inner side it
forms xylem and parenchyma in alternate patches and inner mass of parenchymatous cells form
conjunctive tissue. After a short while the activity of cambium on inner side changes and above
the xylem it starts forming phloem and then again xylem. The xylem formed earlier has bigger
vessels. Around each vascular bundle is developed sclerenchymatous sheath. The cambium after
sometime alters its activity and forms xylem on the inner side, at those places where it was
previously forming the parenchyma and parenchyma in place of xylem. Similar to earlier case
again by change in activity it forms a ring of vascular bundles. Activity of cambium goes on
changing regularly and more rings of vascular bundles are formed. Cork cambium is formed
below hypodermis forms cork in normal fashion (fig.15.11).

Fig.15.11: T.S. Dracaena stem showing abnormal secondary thickenings.

2. Abnormal growth from normal cambium (Secondary Thickening in Boerhaavia Stem:
Dicot):
The anamolous secondary growth of dicot (Boerhaavia) stem showed following tissues. Single
layered epidermis consists of small, radially elongated cells. Cortex is well differentiated and
consists of few layered collenchymatous hypodermis followed by chlorenchyma of 3 to 4 cells
deep, but generally below stomata only single layered. Chlorenchyma is present inner to
collenchyma in the form of 3 to 7 layers with thin walled, oval, full of chloroplasts and enclose
many intercellular spaces. Endodermis is clearly developed and made up of many, tubular,
thickwalled cells. Inner to the endodermis parenchymatous pericycle is present but at some
places it is represented by isolated patches of sclerenchyma. conjoint, collateral and endarch
vascular bundles are present in three rings with two innermost large bundles; in the middle ring
the number ranges from 6 to 14 while the outermost ring consists of 15 to 20 vascular bundles.
The innermost and middle rings are medullary bundles. Vascular bundles of inner and middle
rings may show a little secondary growth. Outermost ring of the vascular bundles contains inter-
fascicular cambium which is absent in other two rings. Cambium develops secondarily from the

pericycle and becomes active. It cuts secondary phloem towards outer side and secondary xylem
towards inner side. Due to these changes the primary phloem becomes crushed and present next
to pericycle. Primary xylem is situated near the pith. Interfascicular cambium also soon becomes
active and cuts internally the row of cells which become thick walled and lignified and are
known as conjunctive tissue. Pith is well developed, parenchymatous and is present in the centre
(fig. 15.13).
Fig. 15.13: Secondary Thickening in Boerhaavia Stem (A) T.S. of stem (B) Detail of a portion.
15.4 SUMMARY
1. In plants increase in size and length of stems and roots is called as growth.
2. Plants are capable to continue their growth throughout their life span.
3. The plant part or cells which are responsible for growth and repair are meristem and such
cells are called meristematic cells.
4. Meristem is of undifferentiated mass of cells, capable of divide continue.
5. Apical meristems are found at the tip or apex and responsible for growth in length or height,
which is known as primary growth.

6. Apical meristems differentiate into the three types of basic meristematic tissues the
protoderm, ground meristem, and procambium.
7. Primary growth in plants occurs at the apical meristem which causes the increase of stem
length.
8. Primary growth occurs in both directions; when plants grow upward or toward the
sunlight necessary for photosynthesis and also downward or geotropically to sink roots
deep into the soil to anchor absorb water and nutrients.
9. Secondary growth in plant occurs when stems or branches grow outward by activity of
lateral meristem.
10. Thus the growth in width or circumference of trees or increases in girth is termed as
secondary growth and it arises from cambium.
11. Dicots and Gymnosperms posses cambium thus secondary growth is possible only in
these plant groups.
12. Monocots, however, do not experience secondary growth due to lack of cambium.
13. Exceptionally in some monocots such as Palm, Yucca, Dracaena, Smilax, Agave,
Coconut etc, secondary growth takes place.
14. Generally cambium function normally and plant grows normally in dicots.
15. Sometimes behavior of cambium changes and causes unequal growth pattern which is
called abnormal secondary growth.
16. There may be various factors which causes abnormal growth in plants.
17. In Several dicots secondary growth deviates considerably from the normal secondary
growth such secondary thickenings are called abnormal or anomalous.
18. The reasons are responsible for abnormal secondary growth in dicots are anomalous
position of vascular cambium, abnormal activity of vascular cambium, formation of
accessory vascular cambium, formation of extrastelar cambium, formation of interxylary
phloem etc.

19. The anamalous secondary growth can be divided in two types: 1. Abnormal growth from
abnormal cambium in monocots 2. Abnormal growth from normal cambium in dicots.
20. In monocots vascular bundles scattered in the ground tissue, but in some cases like
Dioscorea vascular bundles found arranged in two rings around the pith anomously.
15.5 GLOSSARY
Apical meristem: Embryonic, totipotent tissue in the tips of the roots and shoots of plants.
Cambium: A lateral meristem that produces secondary growth.
Conjoint: Vascular bundles which contain both xylem and phloem are called conjoint vascular
bundles.
Cork: A plant tissue composed of cells whose walls are impregnated with suberin, produced by
the cork cambium.
Cork cambium: A narrow cylindrical sheath of meristematic cells that produces cork cells.
Dicot: The plants, that has a pair of leaves, or cotyledons, in the embryo of the seed.
Hypodermis: The tissue immediately beneath the epidermis of a plant especially when
modified to serve as a supporting and protecting layer.
Interxylary phloem: The presence of phloem strands embedded within the secondary xylem (wood).
Intraxylary phloem: Phloem situated on the inner side of the vascular bundles.
Ground tissue: A tissue consisting mostly of parenchyma cells that makes up the bulk of a
young plant.
Meristem: Aformative plant tissue usually made up of small cells capable of dividing.
Monocot: The plants,that has a single cotyledon, in the embryo of the seed.

Phloem: Photosynthate conducting tissue of vascular plants.
Periderm: A tissue primarily consisting of cork cells; outer bark.
Pith: The central parenchymatous tissue in a vascular plant axis.
Primary growth: Growth in length, controlled by the apical meristem.
Sclerenchyma: Tissue, when mature, is composed of dead cells that have heavily thickened
walls containing lignin and a high cellulose content.
Secondary growth: Growth in width initiated and maintained by the vascular cambium and
cork cambium.
Secondary xylem: Xylem produced by the vascular cambium.
Vascular bundle: A strand of tissue composed mostly of xylem and phloem.
Vascular cambium: A lateral meristem that produces secondary vascular tissue in stems and
roots.

1. Primary growth is:

(a) Increase in girth (b) Increase of stem at apex
(c) Formation of cambium (d) All of above
2. Tissue responsible for primary growth is:
(a)Cambium (b) Apical meristem
3. Cambium is:
(a) Permanent tissue (b)Meristematic tissue
(c) Apical meristem (d) None of above
4. Tissues having dividing capability is called:

(a) Permanent (b) Non Meristematic

(c) Abortive (d) Meristematic
5. Primary tissue has:
(a) Dividing capability (b) Differentiation capablity
(c) Both a and b (d) None of above
6. Secondary growth is the feature of:
(a) Monocots (b) Cryptogams
(c) Dicots (d) All
7. Monocotyledons shows secondary growth:
(a)Triticum, agave (b) Yucca, Dracaena
(c) Triticum, smilax (d) none of above
8. Scattered vascular bundles are found in:
(a) Hibiscus (b) Peperomia
(c) Malva (d) All
9. Cork is produced by:
(a) Cambium (b) Cork Cambium
(c) Vascular Cambium (d)All
10. Monocots in which secondary growth occurs:
(a) Hordeum, (b) Dracaena
(c) Both a&b (d) None

(1) The plant cells which have capability of division called_________________ cells.
(2) Dicots and gymnosperms posses _________thus secondary growth is possible only plants of
these two groups only.
(3) Monocots do not experience ______________growth due to lack of cambium.
(4) ____________growth in plants occurs at the apical meristem which causes the increase of
stem length.
(5) Sequential (successive) ring of vascular cambium found in ______________stem.

(6) In Dioscorea vascular bundles found arranged in _______rings around the pith anomously.
(7) Four cortical bundles at each corner is feature of_____________Stem.
(8) In Calotropis phloem is found in xylem strands called _________phloem.
(9) Exceptionally in some monocots such as Palm, Yucca, Dracaena, Smilax, Agave, Coconut
etc, _____________growth takes place.
(10) _________________is of undifferentiated mass of cells, capable to divide continue.

(1) Secondary growth do not found in monocots.
(2) Dicots and gymnosperms lack cambium.
(3) Cambium and cork cambium are responsible for secondary growth.
(4) In Dioscoria ring arranged vascular bundles found.
(5) Dracaena stem shows abnormal secondary growth.
(6) The meristem situated at the bases of internodes is called apical meristem.
(7) Sequential (successive) ring of vascular cambium found in Calotropis stem.
(8) Monocots such as Yucca, Dracaena, Smilax, Agave shows secondary growth.
(9) The two important factors for secondary growth are the vascular cambium and the cork
cambium.
(10) Cambium is the characteristic feature of monocots only.

(1) Define growth.
(2) What is cambium?
(3) Define secondary growth.
(4) What means abnormal secondary growth?
(5) Define included phloem.
(6) What is cork cambium?
(7) What is apical meristem?
(8) Define medullary bundle.
(9) Describe cortical bundles.
(10) Define meristem.

15.6.1 Answer keys: 1-b, 2-b, 3-b, 4-d, 5-c, 6-c, 7-b, 8-b, 9-b, 10-a.
15.6.2 Answer key: 1- meristematic, 2- cambium, 3- secondary, 4- primary, 5- Boerhaavia,
6- two, 7-Nyctanthes, 8- included, 9- secondary, 10- meristem.
10-False.
15.7 REFERENCES
 Cutler D.F., Botha, T. and Stevenson, D.W. (2008) Plant Anatomy, An Applied Approach.
Oxford: Blackwell Publishing.
 Esau, K. & Cheadle, V.I. (1969). Secondary growth in Bougainvillea. Annals of Botany (N.
S.) 33: 807-819.
 Ewers, F.W. (1982). Secondary growth in needle leaves of Pinus longaeva (Bristle-cone
pine) and other conifers: Quantitative data. American Journal of Botany 69: 1552-
1559.
 Cutler D.F., Botha, T. and Stevenson, D. W. (2008) Plant Anatomy, An Applied Approach.
Oxford: Blackwell Publishing.
 Esau, K. 1977. Anatomy of Seed Plants. John Wiley Sons. Inc., New York.
 Evert R.F. and Eichhorn S.E. (2013) Raven Biology of Plants (8th eds,). W.H.
Freeman/Palgrave Macmillan.
 Fahn, A. (1967). Plant Anatomy. Pergamon Press, Oxford.
 Foster, A.S. (1951). Practical Plant Anatomy. Van Nostrand, Princeton.
 Singh, V., Pande, P.C. and Jain, D.K. (2013). Structure Development and Reproduction
in Angiosperms. Rastogi Publications, Meerut.
 Vasistha, P.C. (1968). Plant Anatomy. Pradeep Publication & Co., Chandigarh.

(1) Write short note primary growth.
(2) Describe cambium.
(3) What do you understand by cortical bundles?

(4) Describe cork cambium.

(5) Differentiate between primary and secondary growth.
(6) Differentiate between medullary bundles and cortical bundles.
(7) Write short note incuded phloem.
(8) What do you understand by anomalous secondary growth?
(9) Write a note on abnormal structures in Dracaena stem.
(10) Describe meristem.
(1) Write a detailed note plant growth.
(2) Describe abnormal secondary growth with suitable examples.
(3) Write a note on medullary, cortical bundles and included phloem.
(4) Write a note on abnormal structures of Nyctanthes stem with labeled diagrams.
(5) Describe anomalous secondary growth in Dracaena and Boerhaavia stem with labeled
diagrams.

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MSCBOT-510

Phone No. 05946-261122, 261123

Prof. Lalit Tewari Dr. Hemant Kandpal

Dr. Pooja Juyal

Prof. R.C. Dubey Prof. P.D. Pant

Dr. Pooja Juyal

Dr. S.N. Ojha

Unit Written By: Unit No.

1. Dr. Manish Tripathi, 1, 2, 3 & 4

3. Dr. Manish Dev Sharma 7, 9, 11 & 12

Cheif Course Editor

Dr. Prabha Tewari

Dr. S.N. Ojha Dr. Pooja Juyal

Dr. Kirtika Padalia Dr. Prabha Dhondiyal

Dr. Pushpesh Joshi

Course Title and Code : LABORATORY PRACTICAL-II (MSCBOT-510)

Published By: Uttarakhand Open University, Haldwani, Nainital-263139

Unit-6- Problems based on Genetics 88-116

Unit-7-Identification of Indian varieties of important crops 117-145

BLOCK-3 PLANT DEVELOPMENT PAGE NO.

Unit-10-Study of alternate and distichous, alternate and superposed, opposite 181-193

Unit-11-Microscopical examination of vertical section of leaves 194-218

BLOCK-4 PLANT REPRODUCTION PAGE NO.

Unit-15-Study of the primary and secondary abnormal growth in plants 279-297

After reading this unit students will be able-

Fig.1.1. An overview of the eukaryotic cell cycle

Fig.1.2. The events of eukaryotic cell division as seen under a microscope

1.3 IDENTIFICATION OF DIFFERENT STAGES OF MITOSIS

1.4 STUDY OF MORPHOLOGY OF METAPHASE

1.7 SELF ASSESSMENT QUESTIONS

1.9 SUGGESTED READINGS

1.10 TERMINAL QUESTIONS

2 ZYGOTENE Homologous chromosomes begin to pair.

3 PACHYTENE Homologous chromosomes are fully paired.

4 DIPLOTENE Homologous chromosomes separate, except at

6 METAPHASE 1 Paired chromosomes align on the equatorial

7 ANAPHASE 1 Homologous chromosomes disjoin and move to

8 TELOPHASE 1 Chromosome movement is completed and new

13 Cytokinesis The haploid daughter cells are separated by

2.6 SELF ASSESSMENT QUESTIONS

2.8 SUGGESTED READINGS

2.9 TERMINAL QUESTIONS

UNIT-3 STUDY OF MITOTIC INDEX FROM SUITABLE

3.3 STUDY OF MITOTIC INDEX FROM SUITABLE PLANT

2 Prophase Condensed chromosomes inside a nuclear

3 Metaphase Condensed chromosomes present along

5 Telophase Two nuclear regions present within a

Where, n = Number of cells undergoing mitosis

3.6 SELF ASSESSMENT QUESTIONS

2. In which of these cells mitotic index value should be higher

3. Mitotic index calculation in respect of plants depict that

4. Mitotic activity increases in plants when

3.8 SUGGESTED READINGS

3.9 TERMINAL QUESTIONS

 To prepare semi-permanent and permanent slides of different plant materials.

4.4 TECHNIQUES OF PREPARATION OF PERMANENT SLIDES

Bouin’s fluid 70% Alcohol

UTTARAKHAND OPEN UNIVERSITY Page 52

UTTARAKHAND OPEN UNIVERSITY Page 53