MOLECULAR BIOLOGY AND BIOTECHNOLOGY MSCBOT 604

MSCBOT 604

MOLECULAR BIOLOGY AND BIOTECHNOLOGY

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

UTTARAKHAND OPEN UNIVERSITY

MOLECULAR BIOLOGY AND BIOTECHNOLOGY MSCBOT 604

Expert Committee
Prof. J.C. Ghildiyal Prof. G.S. Rajwar
Retired Principal Principal
Government PG College, Karnprayag Government PG College, Augustmuni

Prof. Lalit M. Tewari Dr. Hemant Kandpal

Department of Botany School of Health Science
DSB Campus, Kumaun University, Nainital Uttarakhand Open University, Haldwani

Dr. Pooja Juyal

Department of Botany, School of Sciences,
Uttarakhand Open University, Haldwani

Board of Studies
Prof. P.D. Pant Prof. S.S. Bargali
Director, School of Sciences HOD, Department of Botany
Uttarakhand Open University, Haldwani DSB Campus, Kumaun University, Nainital

Prof. Amrita Nigam Dr. S.S. Samant

School of Sciences Retd. Director
IGNOU, New Delhi Himalayan Forest Research Institute (H.P)

Dr. S.N. Ojha Dr. Pooja Juyal

Assistant Professor Assistant Professor (AC)
Department of Botany Department of Botany
Uttarakhand Open University, Haldwani Uttarakhand Open University, Haldwani

Dr. Kirtika Padalia Dr. Prabha Bisht Dhondiyal

Assistant Professor (AC) Assistant Professor (AC)
Department of Botany Department of Botany
Uttarakhand Open University, Haldwani Uttarakhand Open University, Haldwani

Dr. Pushpesh Joshi

Assistant Professor (AC)
Department of Botany
Uttarakhand Open University, Haldwani

UTTARAKHAND OPEN UNIVERSITY

MOLECULAR BIOLOGY AND BIOTECHNOLOGY MSCBOT 604

Programme Coordinator
Dr. S.N. Ojha
Assistant Professor
Department of Botany,
School of Sciences
Uttarakhand Open University, Haldwani, Nainital

UTTARAKHAND OPEN UNIVERSITY

MOLECULAR BIOLOGY AND BIOTECHNOLOGY MSCBOT 604

S. No. Unit written by: Unit no.

Dr. S. N. Ojha
1. Assistant Professor 1&4
Department of Botany
Uttarakhand Open University, Haldwani

Dr. Pushpesh Joshi

2. Assistant Professor (AC) 2
Department of Botany
Uttarakhand Open University, Haldwani

Dr. Arun Khajuria

3. Assistant Professor 3&6
Department of Botany
Cluster University of Jammu, Jammu and Kashmir

Dr. Chandrakanta
4. 5
Department of Botany
M.B.P.G. College, Haldwani

Dr. Sushma Tamta

5. Associate Professor, 7
Department of Botany,
D.S.B. Campus, Nainital

Dr. Varsha Joshi

6. Assistant Professor
8
Department of Botany
Surajmal Agarwal (Pvt.) Kanya Mahavidyalaya, Kichcha

Dr. Pankaj Bhatt

7. Visiting Scientist,
9
Agriculture & Biological Engineering,
Purdue University, USA.

Chief Course Editor

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

UTTARAKHAND OPEN UNIVERSITY

MOLECULAR BIOLOGY AND BIOTECHNOLOGY MSCBOT 604

Co-Editors
Dr. S.N. Ojha Dr. Pooja Juyal
Assistant Professor Assistant Professor (AC)
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 Bisht Dhondiyal

Assistant Professor (AC) Assistant Professor (AC)
Department of Botany Department of Botany
School of Sciences School of Sciences
Uttarakhand Open University, Haldwani Uttarakhand Open University, Haldwani

Title : Molecular Biology and Biotechnology

ISBN No. :
Copyright : Uttarakhand Open University
Edition : 2022

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

UTTARAKHAND OPEN UNIVERSITY

MOLECULAR BIOLOGY AND BIOTECHNOLOGY MSCBOT 604

CONTENTS
BLOCK-1 MOLECULAR BIOLOGY PAGE NO.

Unit-1 Biomolecules- Classification 1-41

Unit -2 Nucleic acids, Protein sorting 42-59

Unit-3 Prokaryotic and Eukaryotic regulation of gene expression 60-80

BLOCK-2 BIOTECHNOLOGY

Unit-4 81-111
Basic concepts, Principles and Scope of Biotechnology

Unit-5 Plant cell, Tissue and Organ culture and Application of 112-140
Plant Tissue culture

Unit-6 Organogenesis and Adventives embryogenesis 141-162

Unit-7 Genetic Engineering 163-231

BLOCK-3 BIOSAFETY AND IPR

Unit-8 Bio-and Food-safety, Intellectual Property Rights and 232-254

Ethical Issues

Unit-9 Biotechnological Application 255-267

UTTARAKHAND OPEN UNIVERSITY

MOLECULAR BIOLOGY AND BIOTECHNOLOGY (MSCBOT-604)

Block-1
Molecular Biology
MOLECULAR BIOLOGY AND BIOTECHNOLOGY (MSCBOT-604)

UNIT-1-Biomolecules: Classification

Contents:
1.1 Introduction
1.2 Objectives
1.3 Classification of biomolecules
1.4 Chemical background of biomolecules
1.5 Carbohydrates
1.5.1 Classification of carbohydrates
1.5.2 Structure and stereochemistry of carbohydrates
1.5.3 Properties of carbohydrates
1.5.4 Functions of Carbohydrates
1.5.5 Example of Carbohydrates
1.6 Lipids
1.6.1 Classification of lipids
1.6.1.1 Based on the chemical composition
1.6.1.2 Based on Fatty Acids
1.6.1.3 Based on requirements by the human body
1.6.2 Properties of lipids
1.6.3 Biological significance of lipids
1.7 Proteins
4.10 1.7.1 Structure of proteins
1.7.2 Classification of proteins
1.7.3 Properties of proteins
1.7.4 Functions of proteins
1.8 Nucleic acids
1.8.1 Structure of nucleic acids
1.8.1.1 Types of nucleic acids and their functions
1.8.2 Functions of nucleic acids
1.9 Summary
1.10 Self-assessment questions
1.10.1 Multiple choice questions
1.10.2 Fill in the blanks
1.11 References
1.12 Suggested readings
1.13 Terminal questions
1.13.1 Short answer type questions
1.13.2 Long answer type questions

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1.1 OBJECTIVES
• After reading this unit you would be able to:
 Introduce and Classify the biomolecules;
 Define and enumerate the carbohydrates, lipids, nucleic acids, and proteins
on the basis of their structure and functions;
 Explain the important functional group and structural features of
biomolecules.
 Describe the importance of biomolecules like carbohydrates, lipids, proteins
and nucleic acids.

1.2 INTRODUCTION
The cells carry out life-sustaining essential functions with the help of several organic molecules
present in them. These organic molecules are referred to as biomolecules. A Biomolecule, also
known as a biological molecule, is a broad term for molecules found in organisms that are
necessary for one or more distinctive biological processes, such as cell division, morphogenesis,
or development. Large macromolecules i.e. proteins, carbohydrates, lipids, and nucleic acids and
small molecules such as primary metabolites, secondary metabolites, and natural products are
examples of biomolecules.
Although biomolecules are organic compounds, they differ from other organic molecules in that
they share the same chemical kinds, reactions, and physical principles. The biomolecules have a
broad range of sizes and structures, and they are involved in a wide range of life functions. They
are made of more than 25 naturally occurring elements, mainly of carbon and hydrogen with
nitrogen, oxygen, sulphur, and phosphorus. Carbon compounds have major involvement in the
formation of biomolecules. They covalently bind with other elements to form several other
compounds. Some biomolecules are considered derivatives of hydrocarbons; they are produced
by replacing hydrogen atoms from functional groups like alcohols, amines, aldehydes, ketones,
and carboxylic groups (Kumar et al., 2016). The present unit introduces you to about
Introduction, classification, chemical nature and biological role of carbohydrate, lipids, proteins
and nucleic acids.

1.3 CLASSIFICATION OF BIOMOLECULES

Based on molecular weight and solubility, biomolecules are divided into two categories:
micromolecules and macromolecules. Micromolecules typically have tiny sizes, molecular
weights between 18 and 800 Da (dalton), and high solubilities. Organic or inorganic compounds
can make up micromolecules (minerals, gases and water, amino acid and nucleotides etc).
Macromolecules are large sized. With the exception of lipids, they have a high molecular weight
of 800–1000 dalton and are poorly soluble. Carbohydrates, proteins, nucleic acids, and lipids are
macromolecules.

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Different biomolecules can be classified as aldehyde, ketones and aromatic compounds as

chemical forms. There are four major classes of biomolecules:

Micromolecules (Small sized) Macromolecules (large sized)

i Sugars Carbohydrates
Ii Fatty acids Fats/lipids
iii Amino acids Proteins
iv Nucleotides Nucleic acids

1.4 CHEMICAL BACKGROUND OF BIOMOLECULES

Biomolecules made up of carbon and hydrogen, including large molecules such as proteins,
polysaccharides, lipids and nucleic acids. Generally these biomolecules known as the derivatives
of hydrocarbons and some of the hydrogen atoms replaced by various types of functional groups
such as hydroxyl, methyl, carbonyl, carboxyl, amino, phosphate and sulfhydryl to formed
different bioorganic molecules or biological molecules (Table 1.1). Numerous biomolecules are
polyfunctional, which means they contain two or more functional groups that can affect how
reactively they interact with one another. Basically functional group of biomolecules contain
carbon, hydrogen, oxygen, nitrgen, phosphorus and sulphur besides these biomolecules also
contain several heterocyclic and homocylic ring in their structure for example indol ring in
amino acid tryptophan, phenanthrene ring in steroids. Pyrole is the basic unit of porphyrins found
in several biomolecules like hemoglobine, chlorophyll etc. while thiophene ring is the part of

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vitamin-biotin. The amino acid histadine contain the ring structure of imodazole. Pyrimidines
and purine are the basic elements of the nucleic acids.
Table 1.1 Some important functional group of biomolecule.

Functional group Properties General feature Example of

bimolecule
Name Structure

Hydroxyl OH Polar, Hydrophilic, Capable of Characterized by Carbohydrates

hydrogen bond interactions presence of H
and O

Carbonyl O Polar, Carbonyl group of Characterized by Carbohydrates

C ketones and aldehydes is central C and O
strongly polar group capable of Double bond to
acting as a hydrogen bond oxygen increases
acceptor the polarity

Carboxyl O Ionized to release H+. Since Characterized by Fatty acids

C OH carboxyl groups can release central C bound
H+ ions into a solution, they are to O and OH
considered acidic. Polar,
Capable of hydrogen bond
interactions

Amino NH2 Capable of hydrogen bond Characterized by Protiens

interactions as a base, accepts presence of N
H+ to form NH3+. Since amino
groups can remove H+ from
solution, they are considered
basic.

Phosphate O Charged, ionizes to release H+. Characterized by Nucleic acids

- P Since phosphate groups can presence of P
O O-
O- release H+ ions into solution,
they are considered acidic.

Sulfhydryl SH Polar Characterized by Coenzyme-A

presence of S

Due to their enormous size, the majority of biomolecules have distinctive 3-dimensional shapes.
The molecule's three-dimensional structure is maintained via a large number of non-covalent
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interactions between its atoms. The structure of a biomolecule is flexible rather than static due to
the weak nature of most noncovalent bonds and interactions between the biomolecule and the
solvent. Biomolecules also exhibit stereochemistry as organic compounds. When the molecule
contain stereogenic (or chiral or asymmetric) carbon then it can exist in two different isomeric
enantiomers or diastereomers forms that have different configurations in space and have different
properties.

1.5 CARBOHYDRATES
In the biological system, carbohydrates are the most abundant class of organic molecules found
on the earth because they are one of the major classes of biomolecules, along with proteins and
lipids. In general, carbohydrates are found in both plants and animals. Carbohydrates are the
form in which the solar energy that is used by algae, certain bacteria, and green plants during
photosynthesis is stored. Everyday foods like rice, bread, potatoes, sugar, soft drinks, jams and
fruit juices, salad, etc. contain them in various forms. Carbohydrate is an organic compound, it
comprises of only oxygen, carbon and hydrogen. Originally it referred to compounds of general
formula Cn(H2O)n. Where n is the number of carbons in the molecule represents carbohydrates.
In other words, the ratio of carbon to hydrogen to oxygen is 1:2:1 in carbohydrate molecules.
So, carbohydrates are hydrates of carbon; technically they are polyhydroxy aldehydes and
ketones. Carbohydrates are also known as saccharides, the word saccharide comes from Greek
word Sakkron which means Sugar. They are the major source of energy in many organisms,
serve to store energy, and are structural components in some organisms.
The carbohydrates are poly functional compounds and contain functional group Alcoholic
hydroxy group (-OH), Aldehyde group (-CHO) and Ketone (>C=O). Thus carbohydrate can be
defined as Polyhydroxyaldehydes such as D-glucose, polyhydroxy ketones such as D-fructose,
and large molecules such as sucrose that produce these compounds on hydrolysis.
1.5.1 Classification and nomenclature of carbohydrates
The carbohydrates are divided into three major classes depending on the number of monomer
units or upon whether or not they undergo hydrolysis and if they do, on the number of products
formed. The simplest carbohydrates in this classification are monosaccharides since they cannot
be divided into smaller carbohydrates. Only one unit (mono-single) makes up these molecules.
Oligosaccharides (oligo- few) consists of 3-20 monosaccharide units. Polysaccharides (poly-
many) are the most abundant carbohydrate found in food, it has more than 20 to 100 or 1000 of
monosaccharide units bound together.
1. Monosaccharides: The monosaccharides are polyhydroxy aldehydes or polyhydroxy ketones
which cannot be decomposed by hydrolysis to give simpler carbohydrates. Examples: Glucose,
Fructose, Galactose etc. On the basis of number of carbon in main chain monosaccharides are
also classified as trioses, tetroses, pentoses, hexoses and heptoses (Table 1.2).

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Table 1.2 Clasification of monosacarides

No. of Emprical Aldose Ketose

Carbon Formula
Atoms Types Example Types Example

3 C3H6O3 Aldotriose Gleceraldehyde Ketotriose Dihydroxyacetone

4 C4H8O4 Aldotetrose Erythrose Ketotetrose Erythrulose

5 C5H10O5 Aldopentose Ribose Ketopentose Ribulose

6 C6H12O6 Aldohexose Glucose Ketohexose Fructose

7 C7H14O7 Aldoheptose Glucoheptose Ketoheptose Sedoheptalose

2. Oligosaccharides: Oligosaccharides (Oligo: few) made of short chains of monosaccharide

units, or residues, joined by characteristic linkages called glycosidic bonds and upon the
hydolysis gives definite number (2-10) of monosaccharide molecules. Disaccharide and
trisaccharide are the names for oligosaccharides with two and three monosaccharide units,
respectively.
(i) Disaccharides: The oligosaccharides containing two monosaccharaides units are called
disaccharide, which yield two monosaccharide’s molecules on hydrolysis. Its molecular formula
is C12H22O11.Examples Sucrose, maltose etc. Sucrose (C12H22O11) is disaccharides because
on hydrolysis it gives one molecule of glucose and one molecule of fructose.
H+
C12H22O11 + H2O C6H12O6 + C6H12O6
Sucrose Fractose Glucose

H+
C12H22O11 + H2O 2C6H12O6
Maltose Glucose

ii) Trisaccharides: The oligosaccharides containing three monosaccharaides units are called
trisaccharide, which yield three monosaccharides molecules on hydrolysis and have molecular
formula is C18H32O16. Example- Raffinose, Melezitose.

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H+
C18H32O16 + H2O C6H12O6 + C6H12O6 + C6H12O6
Raffinose Fractose Glucose Galactose

iii) Tetrasaccharides: The oligosaccharides containing four monosaccharaides units are called
tetrasaccharide, which yield four monosaccharides molecules on hydrolysis and have molecular
formula is C22H42O21. Example- Stachyose.

H+
(C6H10O5)n + H2O nC6H10O6
Starch Glucose
or
Cellulose

3. Polysaccharides: The carbohydrates which have higher molecular weight, which yield more
than 10 monosaccharide units on hydrolysis. Some polysaccharides, like cellulose, are linear
chains; others, such as glycogen, are branched. Example- Starch, Glycogen, Dextrin, Cellulose etc.
H
(C6H10O5)n H2O n C6H12O6
Glucose

In general, polysaccharides are amorphous, insoluble in water, and tasteless, whereas

monosaccharides and oligosaccharides are crystalline solids, soluble in water, and sweet to taste.
These substances are collectively referred to as sugars. Differences between monosaccharides,
oligosaccharides, and polysaccharides are shown in Table 1.3.
Table 1.3 Differences between monosaccharides, oligosaccharides and polysaccharides.
Character Monosaccharides Oligosaccharides Polysaccharides
No. of sugar 1 2-10 More than 10
molecules
Glycoside bond Absent Present Present
Molecular weight Low Moderate High
Taste Sweet Minimally sweet No teste
taste
Solubility in water Soluble Soluble insoluble
Nature Always reducing May or may not be Always non
sugar reducing sugar
Example Glucose, Fructose, Sucrose, Maltose Starch, Cellulose,
Glactose Glycogen

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Additionally, the carbohydrates can be categorised as either reducing sugars or non-reducing

sugars. Reducing sugars are all carbohydrates that may reduce Fehiling's solution and Tollen's
reagents, whereas non-reducing sugars are all other carbohydrates. Reducing sugar has the ability
to convert cupric ions from Benedict's or Fehling solution to cuprous ions by carrying a free
aldehyde (-CHO) or ketonic [RC(=O)R'] group. Other than sucrose, all monosaccharides and
disaccharides (such as lactose, melibiose, cellobiose, and gentiobiose) are reducing sugars.
1.5.2 Structure and Stereochemistry of Carbohydrates
Wedge-and-dash structures and Fischer projections are frequently used to illustrate the chemical
structures of carbohydrates (Fig. 1.1). Numerous stereocenters exist for carbohydrates. For
instance, glyceraldehyde only has one. However, as you get to more complex carbohydrates, you
will see an increase in stereocenters. A glucose molecule has four chiral carbons. This suggests
that the glucose molecule contains a total of 16 stereoisomers. The number of stereoisomers is
equal to 2n, where “n” is the number of chiral centres. Stereoisomers are molecules that are
identical chemically but differ only in their spatial arrangement. Asymmetric (chiral) carbon
atoms are found in all monosaccharides, with the exception of dihydroxyacetone, and they all
contain isomeric forms that are optically active. Aldose carbohydrate glyceraldehyde has two
distinct optical isomers, or enantiomers, called d- and l-glyceraldehyde because it has a chiral
carbon atom in the centre. Enantiomers are paired chiral compounds or steroisomers that are not
mirror reflections of one another. Such compounds have the opposite orientation of all chiral
carbon atoms. By convention, one of these two enantiomers is designated as the D isomer, the
other as L isomer.
HC O HC O CH2OH CH2OH
H OH H OH C O C O
HO H HO H HO H HO H
H OH H OH H OH H OH
H OH H OH H OH H OH
CH2OH CH2OH CH2OH CH2OH

Wedge and dash Fischer projection Wedge and dash Fischer projection
structure structure

D-Glucose D-Fructose
(Polyhydroxy aldehyde) ( y ydroxy ketone)
Pol h

Figure 1.1 Wedge-dash structures and Fischer projections of carbohydrates.

A. Open Chain form of carbohydrates
The D and L notation, which describes the arrangement of the final chiral carbon in the chain, is
used to explain the stereochemistry of carbohydrates. It is used to give the name of molecule by
relating it to glyceraldehyde. Fischer's projection is used to distinguish between D and L
carbohydrates. The carbohydrate is given the D configuration in Fischer's projection if the
hydroxyl group is located to the right of the last stereocenter, and the L configuration in Fischer's

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projection if it is put to the left of the final stereocenter carbon. It simply provides information on
how carbohydrates are configured; it makes no mention of the direction in which plane-polarized
light rotates or the fact that molecules with dissimilar atomic configurations are known as
asymmetrical molecules. The D and L arrangement does not provide absolute stereochemistry,
but the anomeric carbon centre can by comparing its orientation to the glyceraldehyde. The D-L
system is significant because it provides the relative arrangement of the molecules. Enantiomers,
for instance, can be quickly designated using the D- and L- notation. The enantiomer of L-
glucose is called D-glucose. Enantiomers can be quickly identified using the D- and L- notation;
for instance, D-glucose is the enantiomer of L-glucose (Fig. 1.2).
H O H O
C C
HO C H H C OH
H O H O H C OH HO C H
C C
HO C H H C OH
HO C H H C OH
HO C H H C OH
CH2OH CH2OH
CH2OH CH2OH
L-Glycerose D-Glycerose L-Glucose D-Glucose
(L-Glyceraldehyde) (D-Glyceraldehyde)

Figure 1.2 Open chain form of carbohydrates and D, L nomenclature.

Additionally, the molecule has optical activity due to the presence of asymmetric carbon atoms.
When a beam of plane-polarized light is passed through a solution of an optical isomer, it will be
rotated either to the right, dextrorotatory (+); or to the left, levorotatory (-). The direction of
rotation is independent of the stereochemistry of the sugar, so it may be designated D (-), D (+),
L (-), or L (+). The D (-) isomer of fructose is one example of a sugar that occurs naturally.
B. Cyclic form of carbohydrates (Hemi-acetal structure)
Carbohydrates can generate intramolecular (cyclic) hemiacetals because they have carbonyl and
alcohol functional groups. There are no intramolecular cyclic forms for smaller carbohydrates
because they are required to be at least a tetrose in order to do that. A 5-membered ring results
from the cyclization of the -OH on the fourth carbon. A 6-membered ring results when the -OH
is provided by the fifth carbon. The 5-membered rings are known as furanoses, and the 6-
membered rings are known as pyranoses. These names are derived from the analogy with the
widely used heterocyclic chemicals furan and pyran, which contain oxygen. Here is an
illustration of how the ubiquitous sugar D-galactose can take on two distinct cyclic forms
(Fig.1.3).
The majority of monosaccharides with five or more carbon atoms in aqueous solutions have a
ring structure. The ring structure is formed by an intramolecular (within the molecule) reaction
between hydroxyl groups and aldehyde group of aldose or ketone group of ketose. A covalent
bond is formed between the carbonyl group and the oxygen of a hydroxyl group along the chain.
It leads to the formation of cyclic (ring) adducts (product formed due to addition of the reactants)

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that are known as hemiacetal and hemiketals, respectively. These cyclic structures now contain
an additional asymmetric carbon atom and therefore, can exist in two steroisomeric forms. The
asymmetric carbon atom is known as anomeric carbon.

H 1 O
C
2 HO OH
H C OH
O 1 OH 5 O 1 OH
3 5 4 6
HO C H HO
4 2 4 2
HO C H 6 3
5 HO OH HO OH
H C OH 3
OH
6 CH OH Furanose
2 Pyranose

D-Galactose O
O

Furane ring Pyrane ring

Figure 1.3 Cyclization of galactose (produced two different cyclic products)

C. Haworth Projections (α and β anomers)

The cyclic forms of carbohydrates are frequently represented by a certain sort of illustration.
They are referred to as forms or Haworth projections. Basically, a Haworth projection is a cyclic
structure with, traditionally, carbon 1 to the right and the bottom portion of the structure oriented
towards the observer. C1 in a cyclic carbohydrate is called anomeric carbon. This carbon used to
be a carbon of the C=O in the open-chain structure prior to the cyclization. Anomeric carbon is
unique since it has no fixed stereochemistry and can exist in either an α-form or a β-form. The α-
and the β-forms are defined as trans or cis isomers of the cyclic carbohydrates where we look at
the anomeric -OH and the C 5 or C6 for furanoses or pyranoses correspondingly.
Cyclization produces an anomeric carbon (the former carbonyl carbon), which results in the
formation of the sugar's and α and β configurations, such as α-D-glucopyranose and β-D-
glucopryanose. In a Haworth projection formula for the α-configuration, OH (C1) is trans to the
CH2OH (C5) group, but in the α-configuration, OH (C1) is cis to the CH2OH (C5) group. They
are referred to as diastereomers because the forms are not mirror images. Thus
monosaccharaides, differing only in this configuration around the carbon atom to which the
carbonyl group is attached (the anomeric carbon), are called anomers (Fig.1.4).

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6 β 6
CH2OH CH2OH
5 O 5 O
H OH H H
H H
4 1 Anomeric carbon 4 1 Anomeric carbon
OH H OH H
OH H OH OH
3 2 3 2 α
H OH H OH

β-D-Glucopyranose α-D-Glucopyranose

6 β 6 6
CH2OH CH2OH CH2OH
5 O 5 OH 5 O
H OH H H H H
H H H
4 1 4 O 4 1
OH H OH H OH H
OH H OH 1 OH OH
3 2 3 2 3 2 α
H OH H OH H OH
β-D-Glucopyranose D-Glucose α-D-Glucopyranose

Haworth projection formula

Figure 1.4 The α- and the β-forms of Glucopyranoses

D. Glycosidic bonds
Glycosidic linkages combine monosaccharides or longer sugar chains with other
carbohydrates to make disaccharides, oligosaccharides, and polysaccharides. It is a specific
kind of covalent bond. Important disaccharides include lactose (galactose + glucose), sucrose
(glucose + fructose) and maltose (glucose + glucose). The bonds that link sugars are called
glycosidic bonds. These monosaccharide sugar chains have the capacity to make additional
glycosidic linkages with alcohols and amines to yield sugar acetals/glycosides and
nucleosides. Glycosidic bonds between sugars are named according to the numbers of the
connected carbons and also with regard to the position of the anomeric hydroxyl group of the
sugar involved in the bond. An O-glycosidic bond is formed when the anomeric carbon of the
sugar joins up with the oxygen atom in the hydroxyl group of the alcohol. Instead, an N-
glycosidic bond is referred to as such if the anomeric carbon of the sugar forms a link with
the nitrogen atom of an amine. If this anomeric hydroxyl is in the α configuration, the linkage
is an α-bond. If it is in the β configuration, the linkage is a β-bond.
• Example- lactose, forming a glycosidic bond between carbon 1 of β-galactose and carbon
4 of glucose. The linkage is, therefore, a β (1→4) glycosidic bond (Fig.1.5).

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6
CH2OH
6 5 O
CH2OH H OH
5 H
O 4 1
OH O OH H
H H
4 1
OH H 3 2
H H H OH
3 2
H OH
D-Galactosyl1-β (1-4)-D-glucose

Figure 1.5 β (1→4) Glycosidic bond

E. Epimer
Compounds that have the same chemical formula but have different structures are called isomers.
For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the
same chemical formula, C6H12O6 (Fig. 1.6).
• Carbohydrate isomers that differ in configuration around only one specific carbon atom
are defined as epimers of each other.
• For instance, the only structural difference between glucose and galactose, which are
both C-4 epimers, is the location of the -OH group at carbon 4. [Note: The carbons in
sugars are numbered beginning at the end that contains the carbonyl carbon-that is, the
aldehyde or keto group].
• Glucose and mannose are C-2 epimers. However, galactose and mannose are not
epimers-they differ in the position of -OH groups at two carbons (2 and 4) and are,
therefore, defined only as isomers.

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Isomers

1
CHO CHO CHO CHO CH2OH
2
HO C OH H C OH H C OH HO C H C O
3
HO C H HO C H HO C H Cl C H HO C H
4
HO C H HO C H H C OH H C OH H C OH
5
H C OH H C OH H C OH H C OH H C OH
6 CH2OH CH2OH CH2OH CH2OH CH2OH
Talose Galactose Glucose Mannose Fructose

C-2 Epimer C-4Epimer C-2 Epimer

Isomers

Figure 1.6 Epimers of carbohydrate with chemical formula, C6H12O6.

I. Mutarotation
Mutarotation is defined as the changes in specific optical rotation by inter conversion of α and β
form of D-glucose to an equilibrium mixture. Enzymes that accelerate the attainment of this
equilibrium are called mutarotases and can be incorporated in assay reagents in order to speed up
the equilibrium formation.
Mutarotation is the change in the optical rotation because of the change in the equilibrium
between two anomers, when the corresponding stereocenters interconvert. Cyclic sugars show
mutarotation as α and β anomeric forms interconvert.
 The optical rotation of the solution depends on the optical rotation of each anomer and
their ratio in the solution.
 Mutarotation was discovered by French chemist Sir Dubrunfaut in 1846, when he
noticed that the specific rotation of aqueous sugar solution changes with time.
 Sugars in the ring form can exist in two states, one where the C-1 hydroxy group is above
the plane of the ring (β) and one where it is below (α).
 In aqueous solution there is a constant interchange between the various conformations via
the breaking open of the hemi acetal structure and its subsequent reforming.

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Figure 1.7 The figure shows the mutarotation of glucose anomers (Source: Mutarotation 2020).

1.5.3 Properties of Carbohydrates

General properties
• Carbohydrates serve as a source of stored energy, fuel, and metabolic intermediates.
• The structural framework of the genetic material, RNA and DNA, is made up of the
sugars ribose and deoxyribose.
• Proteins and lipids involved in cell interactions are connected to carbohydrates.
• Cell walls of bacteria and plants are made of polysaccharides like cellulose.
• Organic molecules known as carbohydrates are aldehydes or ketones with many hydroxyl
groups.
Physical Properties
• Optical Activity: It is the rotation of plane polarised light that produces (+) and (-)
glucose.
• Steroisomerism: compounds with the same structural formula but different spatial
arrangements. Example: In terms of the penultimate carbon atom, glucose contains two
isomers. They are D- and L-glucose, respectively.
• Diastereoisomers: It is the configurational alterations of glucose's C2, C3, or C4.
Mannose and galactose are examples.
• Annomerism - It is the spatial configuration with respect to the first carbon atom in
aldoses and second carbon atom in ketoses.
Chemical Properties
• Osazone formation: Three moles of phenylhydrazine and one mole of aldose combine to
form a crystalline substance which is known as phenylosazone (Scheme-1).
Phenylosazones are helpful derivatives for identifying sugars since they crystallise easily
(unlike sugars).

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• Benedicts test: Simple carbohydrates can detect using Benedict’s Test The Benedict's test
finds reducing sugars (monosaccharides and some disaccharides) that include free ketone
or aldehyde functional groups.
• Oxidation: Sugars are referred to as reducing sugars because they easily oxidise to form
carboxylic acids. Because aldehydes contain an open C=O link, they are simpler to
oxidise. Ketones, however, cannot be oxidised unless they first tautomerize to generate
an aldose.
• Reduction to alcohols: By using Sodium borohydride (NaBH4), the carbonyl group in
aldoses and ketoses can be converted to 1o and 2o alcohols, respectively. An alditol, a
polyalcohol, is the end result of this process. In both arrangements, the reduction of the
ketoses generates a new chiral centre.

1.5.4 Functions of Carbohydrates

Carbohydrates have a variety of functions such as:
• In many animals, carbohydrates are the principal source of energy and are a quick supply
of power. The glycolysis and Kreb’s cycle breaks down glucose to produce ATP.
• The source of energy storage is glucose. Animals store it as glycogen, while plants store
it as starch.
• Stored carbohydrates act as energy source as an alternative of proteins.
• In the production of proteins and lipids, carbohydrates act as intermediaries.
• Carbohydrates combine with lipids and proteins to produce surface antigens, receptor
molecules, vitamins, and antibiotics.
• Carbohydrates are also involved in detoxification, e.g. glucuronic acids.
• As in the cell walls of plants and microbes, they compose structural and defensive
components.
• They are an essential component of connective tissues in mammals.
• Act as structural elements, such as chitin in insects, cellulose in plants, and
glycosaminoglycans in humans.
• They take part in biological transport, cell-cell communication, and growth factor
activation.
• Fiber-rich carbohydrates aid in preventing constipation.
• Additionally, they aid in immune system modulation.
• Non digestable carbohydrates like cellulose, be used as dietary fibres.
• Constituents of nucleic acid RNA and DNA. E.g. Ribose and deoxyribose sugar.

1.5.5 Example of Carbohydrates

• Monosaccharides: Erythrose, Fructose, Galactose, Glucose, Glycerose, Ribose,
Ribulose,
• Oligosaccharides: Lactose, Maltose, Raffinose, Sucrose, Stachyose.

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• Polysaccharides: The composition, occurrences and functions of some important

polysaccharides are given below (Table 1.4):

Table 1.4 Examples of polysaccharides with occurrences, composition and functions.

Polysaccharide Occurrence Composition Functions
Name
Cellulose Plant cell wall Polymer of glucose Cell wall matrix

Chitin Bodywall of arthropods. In Polymer of glucose Exoskeleton

some fungi also Impermeable to water
Glycogen Animals Polymer of glucose Storage of reserve food

Gums and mucilages Gums - bark or trees. Polymers of sugars and Retain water in dry
Mucilages - flower sugar acids seasons
Hemicellouse Plant cell wall Polymer of pentoses and Cell wall matrix
sugar acids
Heparin Closely related to chrondroitin Connective tissue cells Anticoagulant

Hyaluronic acid Connective tissue matrix, Polymer of sugar acids Ground substance,
Outer coat of mammalian eggs protection
Inulin In roots and tubers Polymer of fructose Storage of reserve food

Lignin Plant cell wall (dead cells like Polymer of glucose Cell wall matrix
sclerenchyma)
Murein Cell wall of prokaryotic cells Polysaccharide cross Structural protection
linked with amino acids
Pectin Plant cell wall Polymer of galactose and Cell wall matrix
its derivatives

1.6 LIPIDS
Lipids are a heterogeneous group of organic molecules that are hydrophobic and are structurally
made up of a chain of hydrocarbons (–CH2–CH2–CH2–CH2–). Typically, these are fats, oils,
steroids, waxes, or substances that are similar. The term "Lipid" was coined in 1943 by German
chemist Bloor (the Greek word "lipos" meaning "fat"). It refers to a diverse class of biomolecules
relatively insoluble in water but soluble in organic solvents such as chloroform, ether and
benzene. Chemically, lipids are more diverse than other biomolecules and comprise various
types of esters of different alcohols. They are mainly involved in energy storage and membrane
structure. Lipids can be categorised into a number of different groups. The majority of lipids
serve as molecules that store energy or as parts of the structure of membranes. Some are also
pigments, vitamins, and harmones.

1.6.1Classification of lipids

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Henri Braconnot divided lipids into the two categories of solid grease and fluid oil for the first
time in 1815. However, T. P. Hidlich, who split the simple lipids into grease and waxes,
provided the correct categorization in 1947. Lipids can be classified in four ways, depending on
chemical composition, fatty acids, requirements and sources.

1.6.1.1 Based on the Chemical Composition

On the basis of chemical composition lipids are mainly classified into three types: Simple,
Complex, and Derived lipids.
1) Simple Lipids:
Simple lipids are esters of fatty acids with different alcohols. These are triglycerides, esters of
fatty acids, and wax esters. The hydrolysis of these lipids gives glycerol and fatty acids. Neutral
fats or Triacylglycerols are esters of fatty acids with glycerol, whereas waxes are esters of fatty
acids (saturated or unsaturated) with high molecular weight.
R-OH + R’-COOH R’CO-OR + H2O
Alcohol Acid Ester Water
A. Fatty acids
Fatty acids are the simplest form of lipids. They are a long chain of hydrocarbons (4 to 36
carbons long) with one carboxyl group (Kumar & Mina, 2016). Fatty acids are amphipathic,
having both polar and nonpolar ends. The alkyl chains present in their structure can either be
saturated or unsaturated (Kumar & Mina, 2016). Most naturally occurring fatty acids are
composed of even number of carbon atoms since their biosynthesis requires a concatenation of
C2 units. Most commonly found fatty acids have carbon atoms between 16 and 20. In higher
plants and animals, half of the fatty acids are polyunsaturated. Predominantly found saturated
fatty acids (Fig. 1.8) include Palmitic acid (C16), Stearic Acid (C18) and Arachidic acid (C20).
Based on the hydrocarbon, fatty acid chains are given systematic names The notation used to
represent unsaturated fatty acids is C: n where C represents the number of carbon atoms and n
represents the number of double bonds in the fatty acid. Numbering begins with the first carbon
(carboxy terminal). The 2, 3, and 4 carbons next to the carboxyl carbon are referred to as α, β
and γ carbons, respectively. If the hydrocarbon chain is monounsaturated, the unsaturated chain
is given the suffix "enoic acid" after the name, while the saturated chain is given the suffix
"anoic acid" or "anoate". In the unsaturated fatty acids, the double bonds are always found in the
cis configuration. When there are many double bonds, as in polyunsaturated fatty acids, the
prefix di, tri, etc. is used before the word "enoic acid." One or more double bonds can be found
in unsaturated fatty acids (Fig. 1.8), as in Oleic acid (18:1) , Linoleic Acid (18:2), Linolenic acid
(18:3) and Arachidonic Acid (20:4).

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i) Monoenoic acids: Unsaturated fatty acid having a single double bond.

e.g., 18:1(Δ9) Oleic acid (C17H33-COOH)
CH3-(CH2)7-CH=CH-(CH2)7-COOH
ii) Dienoic acids: Unsaturated fatty acids having two double bonds
e.g., 18:2(Δ9, 12) Linoleic acid (C17H31-COOH)
CH3-(CH2)4-CH=CH-CH2-CH=CH-(CH2)7-COOH
iii) Trienoic acids: Unsaturated fatty acids having three double bonds
e.g., 18:3(Δ9, 12, 15) Linolenic acid (C17H29-COOH)
CH3-CH2-CH=CH-CH2-CH=CH-CH2-CH=CH-(CH2)7-COOH
iv) Polyenoic acid: Unsaturated fatty acids having two or more double bonds, otherwise
known as polyunsaturated fatty acids (PUFA)
E.g. 20:4(Δ5, 8, 11, 14) Arachidonic acid (C19-H31-COOH)
CH3-(CH2)14-(CH=CH-CH2)3-CH=CH-(CH2)3-COOH.
A number of conventions utilize the symbol Δ to denote the number and position of double
bonds in a hydrocarbon chain for example, 18:0 represents a fatty acid having 18 carbon long
chain with no double bond, while18:1 (Δ9) represents a fatty acid having 18 carbon chain length
and one double bond at position 9 and 18:2 (Δ9, 12) represents a fatty acid having 18 carbon
chain length and two double bonds, one at position 9 and the other at 12 starting from the
carboxylic end.

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Figure 1.8 Saturated and unsaturated fatty acid

B. Triglycerides (TG) or Neutral fat

In both animals and plants, glycerides serve as the primary fatty acid storage form.
Triacylglycerols are tri-esters of glycerol and fatty acids, often known as triglycerides. They
have a hydrophobic character and are nonpolar. They are referred to as neutral lipids because
they are charge-free. Triacylglycerol contains varying lengths of fatty acids that can be saturated
or unsaturated. Triacylglycerols can be divided into two categories: simple and mixed. Simple
triglycerides are those with just one type of fatty acid, while mixed triglycerides are those with
two or more different types of fatty acids (Kumar & Mina, 2016).
Triacylglycerols are fatty acid esters of glycerol that are insoluble in water (Fig. 1.9). The three
fatty acids that are esterified to the three alcoholic groups of glycerol give them their distinctive
characteristics. They act as the cell's energy reserves, because lipids are less oxidised than
carbohydrates and proteins, and more energy is released when the same amount of fat is
oxidised, they are useful as energy reserves in the cell. Additionally, they are stored in anhydrous
form because they are nonpolar, as opposed to carbohydrate polymers like glycogen that bind
twice as much water.

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Figure 1.9 Triacylglycerols (fatty acid esters of glycerol)

C. Waxes
Waxes are long-chain fatty acid and long-chain alcohol esters (Fig. 1.10). They are totally
insoluble in water and solid at normal temperature. They are synthesized through the
esterification of high molecular weight monohydroxy alcohol with long-chain fatty acids. The
popularly known beeswax contains triacontanyl palmitate as a major molecule. The hydrophobic
nature of waxes allows them to function as water repellents on leaves of some plants, feathers,
and cuticles of insects. They also act as energy reservoirs for higher aquatic organisms and
planktons.

Figure 1.10 The molecular structure of beeswax

2) Complex Lipids:
The complex or compound lipids contain some other organic molecules in addition to fatty acids
and glycerols. Phospholipids, glycolipids, and lipoproteins are some of them.
A) Phospholipids
The four elements that make up phospholipids are fatty acids, glycerol or sphingosine,
phosphate, and alcohol bound to phosphate. It contains sphingophospholipids, ether
glycerophospholipids or phosphoglycerides. These compounds have an amphipathic character.
i) Glycerophospholipids: Glycerophospholipids (also known as phosphoglycerides) contain
glycerol, two fatty acid molecules, a phosphate group at C-3 and alcohol attached to it (Fig.
1.11). They are derivatives of phosphatidic Acid. They are the most abundant phospholipids
found in the cell membrane, among all the other phospholipids. Phosphatidic acid is the most
fundamental type of phosphoglyceride. The hydroxyl groups at the C1 and C2 carbons of
glycerol are structurally esterifies with the carboxyl groups of two fatty acid chains, while the
hydroxyl group at the C3 carbon is structurally esterifies with phosphoric acid.
Phosphoglycerides frequently contain serine, ethanolamine, choline, glycerol, and inositol as
their alcohol moieties.

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Figure 1.11 Structure of phosphoglycerides

ii) Sphingolipids: The majority of biological membranes are made up of sphingolipids. These
lipids are derived from the C18 amino alcohols sphingosine and dihydrosphingosine. In a
sphingolipid, the amino group at the C-2 position of sphingosine is connected to a fatty acid
residue that can be saturated or monounsaturated with 16, 18, 22, or 24 carbon atoms via an
amide bond. A phosphodiester or a glycosidic bond connects the C1 position to a polar head
group. Its parent structure consists of ceramide, which is a fatty acid joined to sphingosine via an
amide linkage. One example of sphingophospholipids is sphingomyelin which is a major
constituent of the nervous system in higher animals. The backbone of sphingosine, the structure
of ceramide, and a typical sphingolipid are illustrated in fig. 1.12.

Figure 1.12 Example of Sphingolipids.

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B. Glycolipids
Glycolipids are molecules in which the carbohydrate moiety is attached with glycerol and fatty
acids. The head group of the molecule contains sugar (one or more) connected directly to the
hydroxyl group at C1 of the ceramide moiety. It is the third major class of membrane lipids and
generally found on the extracellular face of eukaryotic cellular membranes, and function to
maintain stability of the membrane and to facilitate cell–cell interactions. Glycolipids can also
act as receptors for viruses and other pathogens to enter cells. Some examples of glycolipids are
cerebrosides, gangliosides, sulfolipids and lipoproteins (proteins covalently linked to lipids).
Cerebroside that has a single sugar moiety attached to ceramide; globoside, having multiple
sugar moiety attached to ceramide; and ganglioside, which is a globoside with the head group
containing one or more residues of N-acetylneuraminic acid (sialic acid).

C. Lipoproteins
The lipid and protein complexes are known as lipoproteins. They support the movement of lipids
produced in one organ to throughout the body, such as phospholipids, cholesterol, and
cholesterol esters. Lipoproteins soluble in the blood are categorized into four groups based on
their densities such as chylomicrons, Very Low-Density Lipoproteins (VLDL), Low-Density
Lipoproteins (LDL) and High-Density Lipoproteins (HDL). High-density lipoprotein (HDL),
often known as "good" cholesterol, and low-density lipoprotein (LDL), sometimes known as
"bad" cholesterol, are the two primary subgroups of lipoproteins.

3) Derived Lipids
Derived lipids are the hydrolyzed forms of simple and complex lipids. Its include fatty acids,
fatty aldehydes, ketone molecules, steroids, hormones and lipid-soluble vitamins.
A. Steroids
Steroids are cyclic lipids and complex derivatives of triterpenes. It consists of four fused rings
called steroid nucleus known as ‘cyclopentanoperhydrophenanthrene’ (CPPP) nucleus (Fig.
1.13). Three of these rings have 6 carbons while the fourth ring has five carbons. It’s a planar
ring system and relatively rigid. Any movement around the C-C bonds in this system is
restricted. Cholesterol is weakly amphipathic in nature as it has a polar hydroxyl group attached
to the 3rd carbon atom and a long non polar aliphatic chain attached to the 17th carbon atom.
Cholesterol is the main sterol which serves as not only an important structural component of

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biological cell membranes but is also a precursor to major steroid hormones in the body. Steroids
regulate many important physiological processes in the body as well as carbohydrate
metabolism.

Figure 1.13 Parent compounds of steroids- A: Phenanthrene; B: Sterane

1.6.1.2 Based on Fatty Acids

Based on the type of fatty acids they contain, lipids are categories into two groups: saturated and
unsaturated fatty acids.
A. Saturated Fatty Acids
There are no double or triple bonds in saturated fatty acids. They are a simple, unbranched, and
linear chain of CH2 groups connected with a carbon-carbon single bond and one carboxylic acid
at its end. In general, they have the formula CH3 - (CH2)n - COOH, where n is the number of
methylene groups.Some examples of saturated fatty acids are lauric, myristic, palmitic, stearic,
behenic, and lignoceric acids.
B. Unsaturated Fatty Acids
Unsaturated fatty acids have one or more double or triple bonds. Therefore, they can either be
monounsaturated or polyunsaturated. Natural fatty acids often have a cis rather than a trans
structure. Only a few fatty acids containing triple bonds are found in nature, and they are
frequently derived from plants, such as stearolic acid. The unsaturated fatty acids are named
referring to the number of carbons they contain with the suffix -anoic (for saturated fatty acids)
and -enoic (for unsaturated fatty acids). For example, stearic acid contains 18 carbons and is
named octadecanoic acid (18:0). Here, 18:0 refers to 18 carbon fatty acids with zero double
bonds. Some examples of monounsaturated fatty acids are palmitoleic acid, oleic acid, gadoleic
acid and some common polyunsaturated fatty acids include linoleic acid, linolenic acid, and
arachidonic acid.

1.6.1.3 Based on Requirements by the Human Body

Essential and non-essential fatty acids are the two categories of lipids that are classified based on
nutritional needs.
A. Essential Fatty Acids
The term "essential fatty acids" refers to fatty acids that our bodies are unable to generate or
syntheses. These fatty acids need to be taken through a diet to fulfill the body’s requirement for
different metabolic functions. Some essential fatty acids are linoleic acid, linolenic acid, and
arachidonic acid.

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B. Non-essential Fatty Acids

Non-essential fatty acids include those lipids that are synthesized by our body. They are not
needed to be taken through any outside food source. Example- palmitic acid, oleic acid, and
butyric acid.
1.6.2 Properties of lipids
Physical Properties
• Lipids are relatively insoluble in water. They are soluble in non-polar solvents, like ether,
chloroform, and methanol.
• Lipid molecules have no ionic charges
• Pure fats and oils are colorless, odorless, and tasteless.
• Lipids have a high energy content and burn calories during metabolism. They act as
electrical insulators and protect nerve axons.
• Lipids are greasy in texture and stored in adipose tissues inside the body
• Lipids can either be present in saturated (having only single bonds) or unsaturated
(having one or more double bonds) structural form.
• Fats contain saturated fatty acids, which are solid at room temperature such as animal
fats. Plant fats are unsaturated and are liquid at room temperatures.
• The melting point of fats depends on the length of the chain of the constituent fatty acid
and the degree of unsaturation.
• Geometric isomerism, also known as cis-trans isomerism, is a property of lipid molecules
caused by the presence of a double bond in the unsaturated fatty acid.
• Fats have insulating properties and are poor heat conductors.
• Emulsification is the process by which a lipid mass is divided into numerous tiny lipid
droplets. Before the fats can be absorbed by the intestinal walls, the process of
emulsification takes place.

Chemical Properties
• Saponification: Saponification is the term for the hydrolysis of lipids (Triglycerides) by
an alkali (NaOH/ KOH) or lipase enzymes. Glycerol and soap-like fatty acid salts are
produced as a result of this reaction.
• Hydrolysis of triglycerides: Triglycerides (neutral lipids) on reacting with water form
carboxylic acid and alcohol (Sagar, 2008)
• Hydrogenation: The breakage of double bonds occurs after the reaction of unsaturated
fatty acids with hydrogen. This turns the molecules into saturated fatty acids.
• Halogenation: Free or combined fatty acids in the reaction with halogens gain double
bonds and cause decolorization of halogen solutions.
• Rancidity: Oxidation and hydrolysis of fats and oil to generate a disagreeable odor – this
is known as rancidity (Sagar, 2008).

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1.6.3 Biological Significance of Lipids

Lipids being a significant biomolecule in organisms, serve a variety of functions. Here is a list of
the major roles that lipids play in living things.
i) Energy storage: Triacylglycerols, often known as triglycerides, are an important source
of energy for both plants and animals and are found in adipose tissues. The complete
breakdown of fatty acids releases about 38 kJ/g (9 kcal/g) caloric content. The enzyme
lipase regulates the lipid breakdown process within the body.
ii) The structural component of the cell membrane: The plasma membrane of cells is
made of a lipid bilayer with proteins embedded in it. Glycerophospholipid molecules that
are amphipathic make up the lipid bilayer. The glycolipids and phospholipids in the cell
membrane serve as structural components of the membrane.
• There are also certain non-glyceride lipids in the cellular membrane, such as
sphingomyelin and sterols, which are important for membrane flexibility.
iii) Chemical Messenger: Different kinds of lipids serve as cellular messengers or signalling
molecules. They activate different signaling pathways either by binding with G-coupled
receptors or nuclear receptors. The following are a few examples of lipid compounds
with signalling roles:
• Diacylglycerol and phosphatidylinositol phosphate: They are involved in calcium-
mediated activation of protein kinase C.
• Estrogen, testosterone, and cortisol: These are hormones that regulate a variety of
processes, including as blood pressure, reproduction, and metabolism.
• Prostaglandins: It is an eicosanoid, involved in inflammation and immunity.
• Phosphatidylserine: It is involved in signaling phagocytosis of apoptotic cells by
exposing themselves to the outer leaflet of the bilayer cell membrane.
• Sphingosine-1-phosphate: It’s a potent messenger molecule, involved in calcium
mobilizing regulations, cell growth, and apoptosis.
iv) Other Functions
• Fatty acid absorption: Phospholipids play an important role in the absorption and transportation
of fatty acids.
• Lipids also play important role as pigments (carotene), hormones (vitamin D derivative
and sex hormone), cofactors (vitamin K), and detergents (bile salt).
• Vitamin carriers: Natural fat-soluble vitamins including vitamin A, D, and E are carried
by lipids.
• Lipids in the subcutaneous layer of the skin act as an insulator and safeguard against the
cold. Additionally, fats have a role in regulating body temperature.
• Prostaglandins stimulate uterine contraction, lower blood pressure, vasodilation,
inflammation, and pain.
• Thromboxanes function as vasoconstrictors and stimulate platelet aggregation.
• Prostacyclins act as antagonists of thromboxanes – it’s a potent vasodilator.
• Leukotrienes play functional roles in chemotaxis, inflammation, and allergic reactions.

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• Antibiotic agent: Squalamine, a steroid from the blood of sharks, has been shown to be an
antibiotic and antifungal agent of intense activity. This seems to explain why sharks rarely
contract infections and almost never get cancer.

1.7 PROTEINS
The term "protein" refers to a class of large biomolecules, or macromolecules, made up of one or
more long chains of amino acid residues and they are known as building blocks of life. The most
prevalent macromolecules inside of cells are proteins. They provide structural support, storage,
transport, movement, cellular communications, and defense against foreign substances. Protein’s
function is highly governed by its stable structure. The stability of a protein's structure heavily
influences how it works. There are four layers in this structure: primary, secondary, tertiary, and
quaternary. The sequence of amino acids connected by peptide bonds makes up the primary
structure. Secondary structure is the local folding of a part of polypeptide, while the tertiary
structure is mixture of α-helix and β-sheets. Quaternary structure is the subunit composition of a
protein. Proteins regulate and catalyze the body chemistry in the form of hormones, enzymes,
immunoglobulin’s etc. In this section, we will have deeper insights into the protein structure,
classification and its importance.

1.7.1 Structure of Proteins

There are only 20 distinct amino acids that can be polymerized to produce linear chains to form
proteins. Proteins are the polymers of L-α-amino acids. They are divided into four categories
based on how they are structurally organized such as primary, secondary, tertiary, and quaternary
(Fig. 1.14).

Figure 1.14 Four structure of a protein (Source: microbenotes.com/protein-structure)

A. Primary structure
The primary structure of a protein is formed by the formation of a peptide bond between amino
acids. Each peptide has its own unique sequence of amino acids (Fig. 1.15). It is held together by
covalent peptide bonds, which are formed during the process of protein biosynthesis or
translation. Post-translational modifications such as disulfide formation, phosphorylations and

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glycosylations are usually also considered a part of the primary structure. As with naming of
peptides, the assignment of positions of the amino acids in the sequence starts at the N-terminal
end. Examples of protein with a primary structure are Hexosaminidase, and Dystrophin.

Figure 1.15 Primary structure of a protein (Source: commons.wikimedia.)

B. Secondary structure
Proteins can be arranged differently by twisting their polypeptide chains, which is known as
secondary structure. It is a folded structure within a polypeptide that is due to the formation of
hydrogen bonds between amide hydrogen and the carbonyl oxygen of the peptide backbone. The
alpha helix and the beta sheet (Fig.1.16), first proposed by Linus Pauling in 1951, are the two
primary kinds of secondary structure seen in proteins. Myoglobin is an illustration of a protein
with a secondary structure.
• α-helix: The most common type of secondary structure of a protein is the α-helix exists in hair
protein, keratin. Various conformations can be assumed for a protein by rotation around single
bonds and rigid peptide bonds. The simplest arrangement of a polypeptide chain is α-helix. The
polypeptide coils around an imaginary axis with side groups protruding out from the helix. The
single turn of the helix is 5.4 Angstrom which is the repeating unit of the α-helix. Every
winding turn in an alpha helix has 3.6 amino acids residues. In α-helix protein, a hydrogen
bond is formed between the N−H group to the C=O group of the amino acid. The alkyl groups
of the alpha-helix chain are not involved in the H bonds but maintain the alpha-helix structure.
Intra-hydrogen bonding stabilizes the α-helix. This bond forms between the first amino acid
and the fourth amino acid. The charge of the side chains can destabilize the helix. All the
amino acid side chains point outward from the helix.
• β-sheet: The β-sheet is zig-zag extended conformation of a polypeptide. Three to ten amino
acids are combined to create a beta-strand polypeptide. Beta sheets are involved in forming the
fibrils and protein aggregates observed in amyloidosis. Alike alpha-helix, the residue hydrogen
bond between the adjacent strands is separate from each other. In this case, the orderly
alignment of protein chains is maintained by intermolecular or intramolecular hydrogen bonds.
The β-sheet structure can occur between molecules when polypeptide chains run parallel (all
N-terminal ends on one side) or antiparallel (neighboring N-terminal ends on opposite sides).
β-Pleated sheets can also occur intramolecularly, when the polypeptide chain makes a U-turn,
forming a hairpin structure, and the pleated sheet is antiparallel. In all secondary structures, the

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hydrogen bonding is between backbone –C=O and H-N-groups. Differences between α-Helix
and β-sheet type of secondary structure of Protein are given below:

Table 1.5 Differences between α-Helix and β-sheet type of secondary structure of Protein
α-Helix β-Sheet
Amino acids exist in the right-handed coiled Amino acids exist in an almost entirely extended
rod-like structure. conformation, i.e. linear or sheet-like structure.
Intramolecular hydrogen bonding forms β-sheets are formed by linking two or more beta
within the polypeptide chain to create a strands by intermolecular hydrogen bonds.
spiral structure.
3.6 amino acid residues are winded to form Three to ten amino acids are combined to form a
an α-helix polypeptide. β-strand polypeptide.
α-helix can be a single chain polypeptide. β-sheets cannot be in a single chain Polypeptide.
There must be two or more beta-strands.
Alkyl groups of α-helix are oriented outside Alkyl groups are oriented both inside and outside
of the helix. of the sheet.
Example: Myoglobin, Haemoglobin and Example: Skin Fibres or Fibroin.
Keratin.

Figure 1.16 Enzyme Carboxypeptidase. The β-pleated sheet portions are shown in blue, the green
structures are the a-helix portions, and the orange strings are the random coil areas (Source:
http://thebiologs.blogspot.com/2016/09/cape-1-proteins.html)

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C. Tertiary structure
It is a three-dimensional conformation that is formed due to the interaction between R-groups
or side chains of the amino acids that make up the proteins. There are a variety of forces at
work to stabilize the polypeptide chain in this final structure, including electrostatic attraction,
disulfide bonds, hydrogen bonds, and ionic interactions (Salt bridges). The disulfide bond is
the covalent bond that is most frequently engaged in stabilizing the tertiary structure of
proteins. Tertiary structures are stabilized by hydrogen bonding between polar groups on side
chains or between side chains and the peptide backbone; electrostatic attractions also called
Salt bridges, occur between two amino acids with ionized side chains. The nonpolar groups
prefer to interact with each other, excluding water from inward regions (Fig.1.17). When the
polypeptides fold in spherical shape, they are called globular proteins while fibrous proteins
have extended conformation. Globular proteins (Enzymes) and fibrous proteins are example
of proteins with a tertiary structure.

Figure 1.17 Tertiary structure of Protein (Source: creative-proteomics.com)

D. Quaternary structure
Quaternary structure forms between two or more polypeptide chains. Each polypeptide chain is
called a subunit. The structures can exist between polypeptide chains that are similar or
dissimilar. Hydrophobic, electrostatic, hydrogen, and covalent cross-links are some of the bonds
that contribute to the formation of these structures. Hemoglobin, DNA polymerase, and ion
channels are a few examples of proteins possessing quaternary structures.
Hemoglobin in adult humans is made of four chains (called globins): two identical α-chains of
141 amino acid residues each and two identical β-chains of 146 residues each. Hemoglobin, the
oxygen carrier of blood, has a quaternary structure of 4 polypeptides: 2α and 2β and hence α2β2.
α and β polypeptides resemble the myoglobin structure. In hemoglobin, each globin chain
surrounds an iron-containing heme unit. Proteins that contain non-amino acid portions are called
conjugated proteins. Prosthetic groups are the non-amino acids that make up a conjugated
protein. The globins in hemoglobin are the sections of amino acids, while the heme units are the
prosthetic groups (Fig. 1.18).

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Figure 1.18 Quaternary structure of hemoglobin (Source: www.toppr.com)

1.7.2 Classification of Proteins

On the basis of their form, composition, and solubility, as well as their biological roles, proteins
are divided into the following three categories.
1. Classification of Proteins Based on Shape Axial ratio, which is
i) Globular or Corpuscular Proteins: The shape of globular used to describe any
proteins is primarily spherical or ovoid and they are compactly structure or shape with
folded and coil-folded. They are usually soluble in water. two or more axes. It is
Examples are insulin, plasma albumin, and globular enzymes. the ratio of the lengths
(or magnitudes) of the
ii) Fibrous or Fibrillar Proteins: These proteins have an axial axes to one another, or
ratio greater than 10, which gives them the appearance of long the longer axis divided
fibres or ribbons. They are primarily present in animals and aren't by the shorter axis.
soluble in water or diluted acid solutions. Fibrous proteins provide
structural support and protection. For instance, keratin, fibroin,
collagen, and elastins.
2. Classification of Proteins Based on Composition and Solubility
i) Simple Proteins
These proteins are composed only one type of amino acid as a structural component, and when
they are broken down by acids, the constituent amino acids are released. They are mostly
globular type of proteins except for scleroproteins, which are fibrous in nature. In terms of
solubility, simple proteins are further divided.
a) Protamines and histones: They have a low molecular weight, a simple structure, water
soluble, and do not coagulate by heat. They are strongly basic in character due to the high
content of lysine, arginine. They are fundamental proteins that are only found in animals.
Example: Protamines- clupine, cyprinine, salmine; Histones - globin and nucleoshistones.

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b) Albumins: They are distributed widely in nature and are mostly seen in seeds. They can
dissolve in both water and diluted solutions of salts, acids, and bases. Example: Leucosine,
legumeline, serum albumin.
c) Globulins: They can be divided into two categories: Pseudoglobulins which are soluble in
water and euglobulins which are insoluble in water. They are coagulated by heat. Example:
Pseudoglobulin, serum globulin, glycinine etc.
d) Scleroproteins or Albuminoids: These are typically found in animals and are also referred to
as animal skeleton proteins. They cannot be dissolved in water or diluted solutions of acids,
bases, or salts.
ii. Complex or Conjugated Proteins or Heteroproteins
These are proteins that are produced from amino acids and other chemicals. These chemical
elements or non-protein compounds attached to simple proteins are referred to as prosthetic
groups. The prosthetic group may be either a metal or a compound. A protein without its
prosthetic groups is called as apoprotein while, a protein molecule combined with its prosthetic
group is called holoprotein. Depending on the type of prosthetic group present, complex proteins
are further classified.
a) Metalloproteins: These are proteins that are connected to various metals. Example: Casein,
collagen, ceruloplasmin, etc.
b) Chromoproteins: These are proteins that are joined to a coloured pigment. Example:
Myoglubin, hemocyanin, cytochromes, flavoproteins, etc.
c) Glycoproteins and Mucoproteins: These proteins have carbohydrates as the prosthetic
group. Example: Glycoproteins- Egg Albumin, serum globulins, serum albumins; Mucoproteins
- Ovomucoid, mucin etc.
d) Phosphoproteins: These proteins are linked with phosphoric acid. Example: Casein.
e) Lipoproteins: Proteins forming complexes with lipids are lipoproteins. Example: Lipovitellin,
lipoproteins of blood.
f) Nucleoproteins: These are compounds containing nucleic acids and proteins. Example:
Nucleoproteins, nucleohistones, nuclein.
g) Derived Proteins: These proteins are synthesised from simple proteins by the use of heat,
enzymes, or chemical reagents; they are not naturally occurring proteins. There are two
categories of derived proteins: primary derived proteins and secondary derived proteins.
• Primary derived proteins: These are protein derivatives in which the size of the protein
molecule has not been significantly changed. Primary derived proteins are classified into three
types - Proteans, Infraproteins and Coagulated proteins. Example: Edestan, coagulated egg-
white.
• Secondary derived proteins: Secondary derived proteins are those that are created by
excessively altering the structure and characteristics of protein molecules. The peptide bonds
gradually break down through hydrolysis. They are further classified into 3 types - Proteoses,
Peptones and Polypeptides
3. Classification of Proteins on Biological Function

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i) Enzymic Proteins: They are the most diverse and highly specialised catalytic proteins.
Example: Urease, catalase, cytochrome C, etc.
ii) Structural Proteins: These proteins support in strengthening or protecting biological
structures. Example: Collagen, elastin, keratin, etc.
iii) Transport or Carrier Proteins: These proteins support the movement of ions and molecules
throughout the body. Example: Myoglobin, hemoglobin, etc.
iv) Nutrient and Storage Proteins: These proteins provide nutrition to growing embryos and
store ions.
v) Contractile or Motile Proteins: These proteins function in the contractile system. Example:
Actin, myosin, tubulin, etc.
vi) Defense Proteins: These proteins defend against other organisms. Example: Antibodies,
Fibrinogen, thrombin.
vii) Regulatory Proteins: They regulate cellular or metabolic activities. Example: Insulin, G
proteins, etc.
viii) Toxic Proteins: These proteins hydrolyze or degrade enzymes. Example: Snake venom,
Ricin.

1.7.3 Properties of Proteins

General properties
• The most significant biomolecules are proteins, which are also the building blocks of a
cell's cytoplasm. They are organic substances made up of nitrogen and also, oxygen,
carbon and hydrogen.
• Proteins are the structural elements of body tissues and are made up of amino acids.
• Proteins are referred to as the "bricks" of the body since they form the bones, muscles,
hair, and other tissues.
• A protein's molecular weight ranges from 5 to 300 kilo-Daltons.
• Proteins help the body produce and repair tissues while also providing heat and energy.
• Proteins, like enzymes, are useful substances that participate in metabolic processes.
• Proteins are also used to create antibodies and blood haemoglobin.
Physical Properties
• The shapes of proteins can range from short fibrillar structures to simple crystalloid
forms.
• Proteins can be arranged in two different ways: as globular proteins or fibrous or fibrillar
proteins. The spherical proteins known as globular proteins are found in plants. Fibrillar
proteins are thread-like, they occur generally in animals.
• Proteins are flavourless and colourless, and they have a very large size, which results in v
arious colloidal features.
• Proteins often have high molecular weights between 5 X 103 and 1 X 106.
• They are homogeneous and crystalline.
• Proteins diffuse very slowly and exhibit the Tyndall effect.

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• Proteins tend to change their properties like denaturation. Coagulation frequently occurs
after denaturation. Physical or chemical agents may cause denaturation. Chemical agents
are things like X-rays, radioactive radiation, and ultrasonic waves, while physical agents
include things like shaking, freezing, heating, etc.
• Proteins, like amino acids, have an amphoteric characteristic, meaning they can behave as
both acids and alkalies. Since the proteins are amphoteric by nature, depending on the net
charge, they can form salts with both cations and anions.
• The solubility of proteins depends upon the pH. Lowest solubility is seen at isoelectric
point, the solubility increases with increase in acidity or alkalinity.
• All the proteins show the plane of polarized light to the left, i.e., laevorotatory.
Chemical Properties
• Amino acids are produced as their hydrochlorides when proteins are hydrolyzed by acidic
substances such as concentrated HCl.
• Proteins when are hydrolyzed with alkaline agents leads to hydrolysis of certain amino
acids like arginine, cysteine, serine, etc., also the optical activity of the amino acids is
lost.
• Proteins with reaction with alcohols give its corresponding esters. This process is known
as esterification.
• Amino acid reacts with amines to form amides.
• When free amino acids or proteins are said to react with mineral acids like HCl, the acid
salts are formed.
• Xanthoproteic test: When proteins are boiled with concentrated HNO3, a yellow colour
appears because to the presence of the benzene ring.
• Folin's test is a particular test for tyrosine amino acid, where blue colour develops with
phosphomolybdotungstic acid in alkaline solution due to presence of phenol group.

1.7.4 Function of Proteins

Proteins are vital parts of all living things. It takes part in practically all cellular processes.
Cell signalling, metabolic reaction catalysis, cellular and tissue structure formation, DNA
replication, and molecule transport are all activities in which it participates. Based on their
functional characteristics, proteins are divided into the following classes.
1. Enzymes: The term "biological catalysts" refers to these globular conjugated proteins. Some
proteins act enzymes, they catalyze metabolic reactions by reducing the activation energy
that increases the rate of the reaction. Some examples of protein enzymes are DNA
polymerase, Lysozyme, Nnitrogenase, Lipase, Pepsin and Tripsin.
2. Hormones: - Proteins can act as hormones and control a variety of bodily processes. These
are long polypeptides made up of long chains of linked amino acids. They are important
regulators of the body's physiological functions, such as those related to reproduction,
growth and development, electrolyte balance, sleep, etc. Some examples of these hormones

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are growth hormone (GH), follicle-stimulating hormone (FSH), Insulin hormone is a protein
and it regulated the blood sugar level.
3. Structural proteins: These proteins are rigid and insoluble in water; they are fibrous
proteins. They form the structural component of connective tissues, bones, tendons,
cartilages, nails, hairs, and horns. Examples of structural proteins are Collagen, Elastin, and
Keratin.
4. Respiratory pigments: These pigments are globular proteins, typically soluble in water.
Examples include haemoglobin, which carries blood to all tissues and organs through the
blood, and myoglobin, which delivers oxygen to the working muscles.
5. Transport proteins: Proteins transport different substances in blood of different tissues.
Example: Haemoglobin is an oxygen transport protein. These are structural components of
the cell membrane. They form channels in the plasma membrane to transfer selective
molecules inside the cells. Some of them also form components of blood and lymph in
animals. Examples of transport proteins are Serum Albumin, which transport hemin and
fatty acids; Channel proteins; Carrier proteins and Fibrinogen (a glycoprotein helps in
healing of wounds. It prevents blood loss and inhibits passage of germs).
6. Motor proteins: These proteins are involved in the contraction and relaxation of the muscle
(muscle movement). It includes Actin, Myosin, Kinesin, and Dynein.
7. Storage proteins: These proteins are the storage reserve of amino acids and metal ions in
cells. They are present in eggs, seeds, and pulses. Examples of storage proteins include
Ferritin, Ovalbumin, and Casein.
8. Toxins: These proteins are generally produced by bacteria. They include Diphtheria toxin,
Pseudomonas exotoxin, and ribosome-inactivating proteins. They help bacteria to attack and
kill their host organism by creating cytotoxicity.
9. Defense protein: Some proteins act as antibodies; they protect the body from the effect of
invading species or substances.

1.8 NUCLEIC ACIDS

The nucleic acid was first discovered by Friedrich Miesher, a Swiss physician in 1869 isolated
and identified a macromolecular substance from the pus cells in the nuclei of leukocytes and
from salmon sperm which he termed as nuclein. Meischer’s student Richard Altmann in 1899
used the term nucleic acid for phosphorus containing nuclein, which was later proved to be the
hereditary material in 1950s. Later, further studies showed that it’s a mixture of basic proteins
and phosphorus-containing organic acid (Kumar et al., 2016).
Nucleic acids are long-chain polymeric molecules. The monomer or the repeating unit is known
as the nucleotides and hence sometimes nucleic acids are referred to as polynucleotides. Nucleic
acids can be defined as organic molecules present in living cells. It plays a key factor in
transferring genetic information from one generation to the next. Nucleic acids are composed of
DNA-deoxyribonucleic acid and RNA-ribonucleic acid that form the polymers of nucleotides.
You will study in detail about nucleic acids in Unit 02.

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1.8.1 Structure of Nucleic acids

Structurally, nucleic acids (both DNA and RNA) are polymers of nucleotides (or
polynucleotides) composed of pentose sugar, phosphate and nitrogenous bases. Pyrimidines and
Purines are two types of nitrogenous bases. Pyrimidines are composed of cytosine and thymine.
Purines are composed of guanine and adenine. Thymine is replaced by Uracil in ribonucleic acid
whereas deoxyribonucleic acid comprises of all four bases. Sugar is deoxyribose in DNA
(deoxyribonucleic acid) while ribose in RNA (ribonucleic acid). Deoxyribose means the 2'
carbon of sugar lacks the oxygen atom which is present in ribose. Numerals with a prime (2' or
3') distinguish atoms of the sugar from those of the bases. Structure of Ribose and deoxyribose
(pentose sugar) Sugars (Fig. 1.19) in the nucleic acid backbone are linked together by
phosphodiester −bond/linkages/bridges. Phosphate group is attached to C-5 of pentose. During
the formation of a phosphodiester bond, the C-5 of one sugar moiety is joined to the OH of C-3
with phosphate group (phosphate attached to C-5 of pentose). These phosphodiester bonds are
strong covalent bonds and do not break even on heating the DNA molecule at high temperature.

Figure 1.19 Structure of Ribose and Deoxyribose (pentose sugar) Sugars

1.8.1.1 Types of Nucleic Acids and Their Functions

Based on nature, structure, and function, the nucleic acids are categorized into two groups:
Deoxyribonucleic acids (DNA) and Ribonucleic acids (RNA).
A) Deoxyribo Nucleic acids (DNA)
DNAs are the hereditary material that resides inside the nucleus. In 1953, the first structure of
DNA double helix (B-form of DNA) was discovered by Watson and Crick (Fig. 1.20). DNA has
two other forms as well, A and Z forms. The conformation DNA will adopt depends on the
hydration level, DNA sequence, and chemical modification of the bases, the type, and
concentration of a metal ion in the solution. The double helix structure represents two
polynucleotides DNA coiled around a central helix. The two strands are antiparallel and interact
by hydrogen bonds between complementary base pairs. In some cases, like at low pH, the triple
helix form of DNA also exists. It’s formed by laying a third strand into the major groove of the
DNA.

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It is the genetic material that stores all the information required to be transferred to the progeny.
It specifies the biological development of all living organisms
and viruses. Are you aware?
Compared to DNA
duplexes, RNA duplexes
are more stable. RNA
duplexes need a greater
temperature to denaturate
than DNA duplexes do at
physiological pH. However,
the physical cause of this
distinction is still unknown
(Kumar et al., 2016).

Figure 1.20 Double helical structure of DNA (B-form) (Source: theory.labster.com)

Do you know?It is believed that, around 4 billion years ago, RNA was the first genetic material!
Scientists say it is largely because of its self-replicating ability and enzymatic activity. This
hypothetical period is known as the RNA world. But when the protein-forming enzymes came
into existence, DNA became the most dominating and stable form of genetic material. The DNA
structure is more stable than RNA because of the absence of a 2’ hydroxyl group. The other
advantage DNA has is that its double-stranded structure allows for the correction of mutations as
well (Kumar et al., 2016).
B) Ribonucleic acids (RNA)
RNA is present in all living cells. It has different roles to play in different organisms. It acts as
genetic material in some viruses and has
enzymatic activity in other organisms (where Do you know? It is estimated that
it is called ribozyme). Three types of RNA approximately 4 billion years ago, RNA was the
are present among organisms: rRNA, first genetic material! Scientists say it is largely
mRNA, and tRNA. All three have essential because of its self-replicating ability and
roles in the development and maintenance of enzymatic activity. This hypothetical period is
life. known as the RNA world. But when the protein-
The importance of RNA and DNA is forming enzymes came into existence, DNA
incomparable. DNA carrying the genetic became the most dominating and stable form of
information can’t leave its home, the genetic material. The DNA structure is more stable
nucleus, and this is why RNA exists. They than RNA because of the absence of a 2’ hydroxyl
are involved in the transfer of genetic group. The other advantage DNA has is that its
information for protein synthesis via the double-stranded structure allows for the correction
processes of transcription and translation of mutations as well (Kumar et al., 2016).
(outside the nucleus), and they control gene

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expression as well (Kumar et al., 2016). Structurally, RNA exists in both single-stranded
(primary structure) and double-stranded (secondary structure) forms. The double-helical
structure of RNA is present in the A form.

1.8.2 Functions of Nucleic Acids

• Nucleic acid is responsible for the transmission of inherent characters from parent to
offspring.
• DNA fingerprinting is a method used by forensic experts to determine paternity. It is also
used for the identification of criminals. It has also played a major role in studies
regarding biological evolution and genetics.
• The major function of nucleic acids is to store the genetic code of living organisms. DNA
reserves genetic information and is responsible for maintaining the identity of species
over the centuries.
• They are responsible for the synthesis of protein, where RNA functions as an adapter
molecule. RNA facilitates the translation of protein from DNA. In the process of protein
synthesis, mRNA copies DNA and carries the information to rRNA, where rRNA
decodes the information. tRNA takes amino acids to rRNA, where the protein is formed.
• Cellular metabolism is a function of DNA, where it integrates a complex set of
biochemical pathways devoted to the maintenance of cell functions.

1.9 SUMMARY
• A Biomolecule, is a broad term for molecules found in organisms that are necessary for
one or more distinctive biological processes, such as cell division, morphogenesis, or
development. Large macromolecules i.e. proteins, carbohydrates, lipids, and nucleic acids
and small molecules such as primary metabolites, secondary metabolites, and natural
products.
• Based on molecular weight and solubility, biomolecules are divided into two categories:
micromolecules and macromolecules.
• The carbohydrates are poly functional organic compounds, it comprises of only oxygen,
carbon and hydrogen. Its contain functional group Alcoholic hydroxy group (-OH),
Aldehyde group (-CHO) and Ketone (>C=O). Originally it referred to compounds of
general formula Cn(H2O)n.
• The carbohydrates are divided into three major classes depending on the number of
monomer units such as Monosaccharides, Oligosaccharides and Polysaccharides. Glucose
is the main carbohydrate.
• The maltose, sucrose and lactose are common examples of naturally occurring
disaccharides. Starch and glycogen are polysaccharides of glucose in plants and animals.
• Wedge-and-dash structures and Fischer projections are frequently used to illustrate the
chemical structures of carbohydrates. Fischer's projection is used to distinguish between
D and L carbohydrates.

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• Glycosidic linkages combine monosaccharides or longer sugar chains with other

carbohydrates to make disaccharides, oligosaccharides, and polysaccharides. It is a
specific kind of covalent bond.
• Carbohydrates are the principal source of energy and are a quick supply of power. The
Glycolysis and Kreb’s cycle breaks down glucose to produce ATP.
• Lipids are a heterogeneous group of organic molecules that are hydrophobic and are
structurally made up of a chain of hydrocarbons (–CH2–CH2–CH2–CH2–). Typically,
these are fats, oils, steroids, waxes, or substances that are similar.
• On the basis of chemical composition lipids are mainly classified into three types:
Simple, Complex, and Derived lipids.
• Simple lipids are esters of long chain fatty acids and alcohol. They include fats, oils and
waxes. Compound lipids are made up by further modification of simple lipids such as
addition of phosphate, sulfate and carbohydrate or protein group. This class of lipids
includes phospholipids, glycolipids and lipoproteins.
• Derived lipids are derived from hydrolysis of simple and compound lipids. Steroids,
cholesterol, various lipid soluble hormones as well as fatty acids belong to this class of
lipids.
• The term protein refers to a class of macromolecules, made up of one or more long chains
of amino acid residues and they are known as building blocks of life.
• There are only 20 distinct amino acids that can be polymerized to produce linear chains
to form proteins. Proteins are the polymers of L-α-amino acids. They are divided into
four categories based on how they are structurally organized such as primary, secondary,
tertiary and quaternary.
• Amino acids are joined together by peptide bonds to form the basic structure of proteins.
Proteins participate in almost every biochemical process in the body.
• Nucleic acids are one of the major biomolecules required for the proper functioning of
the body. These are the molecules responsible for carrying the genetic information from
parents to offspring, gene expression, and synthesis of proteins required for metabolic
functions.

1.10 SELF ASSESSMENT QUESTIONS

1.10.1 Multiple Choice Questions
1. There are ______ major classes of biomolecules.
a) Four b) Six
c) Three d) Two
2. Biomolecules are very large molecules of many atoms, which are bound together from
a) Ionicaly b) Non-covalently
c) Covalenty d) None of the above
3. Lipids are insoluble in water because lipid molecules are
a) Hydrophobic b) Hydrophilic
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c) Neutral d) Both a & b

4. In double helix of DNA, the two strands are
a) Coiled over base b) Coiled around each other
c) Coiled around a common axis d) Coiled differently
5. Nucleotides are building blocks of nucleic acids, it is composition of
a) Base, Sugar, and Phosphate b) Base and Phosphate
c) Base, Phosphate, and carbon d) Sugar and phosphate
6. ATP is____________
a) Types of nucleotide b) Types of nucleic acid
c) Types of enzyme d) Both a & b
7. Amino acids are mostly synthesized from
a) Fatty acids b) α-ketoacids
c) Proteins d) None of the above
8. In which of the following groups are all polysaccharides?
a) Glycogen, Cellulose and Starch
b) Glycogen, Sucrose and Starch
c) Glycogen, Cellulose and Fructose
d) Glycogen, Cellulose and Glucose
9. Sucrose is also called as ____
a) Milk sugar b) Invert sugar
c) Laevose d) Dextrose
10. Linoleic acid and α-linolenic acid are types of
a) Essential fatty acids b) Simple lipids
c) Complex lipids d) Both a & b
11. DNA polymerase, and ion channels are example of_______________protein
structure.
a) Tertiary b) Secondary
c) Primary d) Quaternary
12. Which of the following are fibrous proteins?
a) Insulin and Albumin b) Salmine and Clupine
c) Fibroin and Elastins d) Casein and Myoglubin
13. Polysaccharides are formed by _______
a) Vanderwaal forces b) Phosphodiester linkage
c) Glycosidic linkages d) Peptide linkage
14. In RNA, thymine is replaced by
a) Cytosine b) Adenine
c) Uracil d) Guanine
15. Glycoside bond are absent in
a) Monosaccharaides b) Oligisaccharides
c) Polysaccharides d) Both b & c

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1.10.2 Fill in the Blanks

a) The alpha helix and the beta sheet first proposed by ……………… in 1951.
b) A glucose molecule has four chiral carbons so that glucose molecule will have a total
of ………………stereoisomers.
c) Palmitic acid, Stearic Acid and Arachidic acid are examples of saturated fatty.
d) Serum albumin is the example of……………… proteins.
e) The double helix structure represents..……..polynucleotides DNA coiled around a
central helix.
f) The disulfide bond is the covalent bond that is most frequently engaged in …………..
the tertiary structure of proteins.
g) A protein without its prosthetic groups is called as ……………..
h) Waxes are long-chain of …………..and alcohol esters.
i) Triacylglycerols are tri-esters of……………………….
j) The α- and the β-forms are defined as ……………...........isomers of the cyclic
carbohydrates.
Answer key
1.10.1: 1-(a); 2-(c); 3-(a); 4-(c); 5-(a); 6-(a); 7-(b); 8-(a); 9-(b); 10-(a); 11-(d); 12-(c); 13-(c); 14-
(s); 15-(a)
1.10.2: a) Linus Pauling; b) sixteen; c) saturated fatty; d) transport; e) two; f) stabilizing; g)
apoprotein; h) fatty acid; i) glycerol and fatty acids; j) trans or cis

1.11 REFERENCES
Aryal, S. (2018). Lipids- definition, properties, structure, types, examples, functions. Retrieved
from https://microbenotes.com/lipids-properties-structure-classification-and-functions/.
Classification of Proteins Based on Structure and Function. Retrieved
from https://www.easybiologyclass.com/classification-of-proteins-based-on-structure-
and-function/#.
Jain, J. L., Fundamentals of Biochemistry, S. Chand & Company Ltd. India.
e-gyankosh, Block-3 Essential Molecules of Biochemistry, Unit-09, Biomolecules,
http://egyankosh.ac.in//handle/123456789/68255.
e-Pathshala, Bio-organic and Biophysical Chemistry, Paper No. : 16; Module No.: 7; Protein
Structure.
e-Pathshala, Bioorganic and Biophysical chemistry, Paper No. : 16; Module No.: 5, Nucleotides
and polynucleotides
e-Pathshala , Bio-organic and Bio-physical Chemistry, Paper No. : 16; Module No.: 2:
Constituents of Cells; Lipids.
Hormones. Retrieved from https://courses.lumenlearning.com/suny-ap2/chapter/hormones/#.
Kumar, Pranav & Mina, Usha. (2016). Life Sciences, Fundamentals, and Practice, Part I.

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Manisha, M. Types of Polysaccharides (3 Types). Retrieved

from https://www.biologydiscussion.com/carbohydrates/polysaccharides/types-of-
polysaccharides-3-types/44929.
Mutarotation (2020). Retrieved
from https://chem.libretexts.org/Ancillary_Materials/Reference/Organic_Chemistry_Glos
sary/Mutarotation
Nelson and Cox, Lehninger, Principles of biochemistry, Fourth Edition.
Orders of protein structure. Retrieved
from https://www.khanacademy.org/science/biology/macromolecules/proteins-and-
amino-acids/a/orders-of-protein-structure.
Overview of Biomolecules. Retrieved from
http://med.fau.edu/students/md_m1_orientation/Overview. pdf.
Protein Toxins. Retrieved from https://www.creative-biolabs.com/adc/protein-toxins.htm#.

1.12 SUGGESTED READINGS

Jain, J. L., Fundamentals of Biochemistry, S. Chand & Company Ltd. India.
Nelson and Cox, Lehninger, Principles of biochemistry, Fourth Edition.

1.13 TERMINAL QUESTIONS

1.13.1 Short Answer Type Questions
1. What is the glycosidic linkage or bond?
2. What do you understand about saturated and Unsaturated Fatty acids?
3. Write a note on the mutarotation.
4. Describe in brief about properties of lipids?
5. Write a short note on classifications of biomolecules.
6. Distinguish monosaccharides, oligosaccharides and polysaccharides.

1.13.2 Long Answer Type Questions

1. What are Biomolecules? Discuss about properties of carbohydrates, proteins and lipids.
2. What are Carbohydrateds? Explain its structure and functions.
3. Give a detailed account on classification, structure and functions of lipids.
4. Write an essay about structure and functions of proteins.

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UNIT 2: NUCLEIC ACIDS, PROTEIN SORTING

Contents:
5.1 Objectives
5.2 Introduction
5.3 Physical and chemical structure of DNA
5.4 Watson and crick model of DNA
5.5 Types of DNA
5.6 DNA as genetic material
5.7 RNA synthesis and its types
5.8 Post Transcriptional Modification
5.9 Genetic code
5.10 Protein sorting
5.11 Summary
1.12 Bibliography
1.13 Suggested readings
1.14 Terminal questions

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1.1 OBJECTIVES
After reading this unit you will be able to:
• Describe physical and chemical structure of DNA and Types of DNA.
• Understand Watson and Crick model of DNA.
• Explain RNA, its synthesis and types.
• Describe Post transcriptional changes in RNA.
• Know about Genetic Code.
• Understand Protein Sorting.

1.2 INTRODUCTION
Cell contains four major classes of biomolecules, i.e. carbohydrates, lipids, proteins, and
nucleic acids, making the majority of cell’s mass. Each biomolecule is an important
component of the cell that performs different type of functions. Among these biomolecules,
nucleic acids, namely DNA or RNA, serve as the ultimate source or storehouse of biological
information in every organism. Nucleic acids, due to its millions of monomeric units, are
large and heavy with molecular weight from 30,000 to several millions. Johann Friedrich
Miescher, in 1969, first isolated a substance from the pus cell, obtained from discarded
surgical bandages. Miescher called this material as ‘nuclein’, which was later renamed as
‘nucleic acid’. Nucleic acid carries all the hereditary information from parents to offspring.
Being the linear polymer of nucleotides, they are also known as polynucleotide. Nucleotides
are made by combining nucleosides, i.e. heterocyclic rings of nitrogenous bases and pentose
sugars, and a phosphate groups. Each nitrogenous base is linked to a sugar to which one
phosphate group is attached. Subsequent studies have shown that there are two types of
nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

DNA or RNA is the principal genetic materials present in entire living organism. DNA is
genetic material of most of the organisms, except some viruses, which have RNA as their
genetic material. DNA is a macromolecule and is a double helical chain of
polydeoxyribonucleotides. Eukaryotic DNA is present in membrane bound cell organelle,
whereas, prokaryotic DNA, lying free in cytoplasm, is known as nucleoid. In prokaryotes it
also occurs as plasmids, both plasmid and nucleoid are double stranded circular DNA. RNA
is a generally single stranded, linear polymer of nucleotides consisting of a ribose sugar and
phosphate backbone (rather than deoxyribose as in DNA). RNA also differs from DNA since
the base, uracil, replaces thymine found in the DNA.

1.3 PHYSICAL AND CHEMICAL STRUCTURE OF DNA

Each nucleotide (a subunit of nucleic acid) comprise of three parts viz. (i) phosphate group,
(ii) sugar component and (iii) heterocyclic nitrogenous bases.
(i) Phosphate group: Phosphoric acid (H3PO4) has three reactive hydroxyl (-OH) groups of
which two are involved in making bond with pentose sugar forming sugar-phosphate back
bone of nucleic acid. In a nucleotide, the phosphate groups binds to C5' and C3' of the ribose
or deoxyribose sugar through sugar-phosphate bonds known as phosphodiester linkages.

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(ii) Sugar: The only two types of pentose sugar viz. ribose and deoxyribose are present in
nucleic acid. The sugar component of RNA is ribose, whereas in DNA it is deoxyribose. The
chemical structure of

(iii) Nitrogenous base: A nitrogenous base is covalently linked to the carbon of the sugar
residue with the help of glycosidic bond. Each nucleic acid contains millions of nitrogenous
bases, but only four different types of bases can occur in a nucleic acid. The nitrogenous bases
found in nucleic acid are planar, aromatic and heterocyclic molecule which are related to
either purine or pyrimidine. Purines (double ring system) include Adenine and Guanine
whereas pyrimidines (single ring system) include Cytosine, Thymine and Uracil.

Purines
Purines are double ringed aromatic organic compound consisting of a six-membered
pyrimidine ring fused to a five-membered ring i.e. imidazole ring. In purine a total of four
nitrogen atoms are present. In first ring of purine, numbering of carbon atoms is done in anti-
clock wise direction whereas in other carbon atoms are numbered in clockwise direction. It is
water soluble and is especially found in high concentration internal organs such as liver and
kidney. Both DNA and RNA contain two types of purines i.e. Adenine and Guanine.

Pyrimidines
Pyrimidines are six membered, single ringed, simple aromatic compounds composed of
carbon and nitrogen atoms. In a pyrimidine ring two nitrogen atoms present at 1st and 3rd
position and four carbon atoms present at 2nd, 4th, 5th and 6th position. The most common
pyrimidine occurring naturally are cytosine, thymine and uracil. The first two, viz. cytosine
and thymine, are present in DNA, whereas in case of RNA uracil is present in place of
thymine.

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Figure: Purines and Pyrimidines

Chargaff’s rule
Erwin Chargaff in 1950 studied the bases and other components of DNA and concluded that
almost all DNA, irrespective of type of organism or tissue, maintain certain properties. He
proposed that DNA of any cell organism should have pyrimidine and purines in 1:1 ratio i.e.
the amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine
(G) is equal to the amount of cytosine (C). The total amount of purines (A + G) is equal to the
total amount of pyrimidines (C + T). He also proposed that adenine (A) always pairs with
thymine (T) with two hydrogen bonds and cytosine (C) always pairs with guanine (G) with
three hydrogen bonds.

Nucleoside and Nucleotide

N of a Nitrogenous bases (N9 in case purine whereas N1 in pyrimidine) attaches to C1 carbon
of pentose sugar with the help of a glycosidic bond. The whole structure formed by this
association is known as Nucleoside. Depending on the type of sugar nucleosides are of two
types: a) Ribonucleoside (if pentose sugar is ribose) and b) Deoxyribonucleoside (if pentose
sugar is deoxyribose). A phosphate group binds to pentose sugar of nucleoside with the help
of phosphodiester bond to constitute a nucleotide. Phosphate group attaches to C5 of the
ribose and C3 of deoxyribose sugar to form ribonucleotide and deoxyribonucleotide
respectively.

William T. Astbury, an experienced crystallographer, studied calf thymus DNA with the help
of X-ray diffraction technique. Astbury missed the actual structure of DNA due to the poor
quality of the photos. During 1950’s 3 separate groups or researchers were intensively
working to discover the structure of DNA: 1) Maurice Wilkins and Rosalind Franklin, 2)
Linus Pauling and 3) James Watson and Francis Crick. Wilkins studied nucleic acid and
proteins via X-ray imaging and successfully isolated fibres of DNA. Rosalind Franklin, an
expert in X-ray crystallography, joined King’s College, London and took the high quality
photographs of DNA, she discovered two forms of DNA: A form (crystalline form) and B

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form (paracrystalline form). In 1953, Pauling along with Corey proposed a triple stranded
helical model of nucleic acid in which phosphate forming the helical core and the bases
placed outside. In 1953 James Watson visited King’s College and was shown Franklin’s
image by Wilkins. Based on the x-ray diffraction image and Chargaff’s rule Watson and
Crick successfully deduced the double helical structure of DNA.

Figure: X-ray crystallography of DNA

1.4 WATSON AND CRICK MODEL OF DNA

J. D. Watson and F. H. C. Crick, in 1953, studied physical and chemical properties of DNA
and deduced a three dimensional model of DNA as double helix. Watson and Crick, on the
basis of Chargaff’s rule and x-ray diffraction image of Wilkins and Franklin, proposed that
DNA is composed of two complementary strands of polynucleotides twisted around each
other in the form of double helix and attached together by hydrogen bonding between purines
and pyrimidine bases. The two polynucleotide strands forming the double helix are anti-
parallel, i.e., both the chains run in opposite direction. If a chain in a helix is aligned in
5’→3’ direction, then the other chain in the helix will be aligned in 3’→5’ direction. A free
OH group is present at 3’ end of a helix and a PO43- at 5’. The phosphate group imparts
negative charge to the DNA molecule. The sugar phosphate backbone of each polymerized
nucleotides strand projects outward the helix and forms the backbone of the structure whereas
the nitrogenous bases toward the center. A pyrimidine base in one polynucleotide chain is
hydrogen bonded with purine base of other chain, i.e. adenine (A) and thymine (T) pair
together, and cytosine (C) and guanine (G) pair together. When adenine occurs in one strand,
thymine is found in the complementary one; similarly, when guanine occurs in one strand,
cytosine is found in the other. The pairing between the bases held both the strands in a helix
together and makes it a stable structure. X-ray diffraction images of DNA have determined
that there are approximately 10 bases per turn in DNA (i.e. it make one complete turn after

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every 10 residues). Two adjacent bases are separated by the distance 3.4 Å, hence the length
of one complete turn of helix is 34 Å. The diameter of the helix is 2 nm. Two grooves of
different width are formed due to asymmetrical spacing of the sugar-phosphate backbones
between the adjacent turns. One wider is known as major groove (ca. 22 Å wide) and the
other narrower is known as minor groove (ca. 12 Å wide). These grooves are locations where
protein binds to DNA.

1.5 TYPES OF DNA

DNA is a flexible molecule. It exist in various three dimensional structure. Early x-ray
diffraction studies by Franklin, Goslin, Wilkins and co-workers revealed the presence of two
kinds, i.e. A form and B form, of DNA. The double helical model of DNA proposed on the
basis of study of physical and chemical property by Watson & Crick is commonly referred as
B form of DNA or B-DNA. In B- form the two polynucleotide strands are spirally coiled
around each other to form right handed helix. A form contains 11 base pair per turn and
similar to B form it also shows right handed coiling. Recent studies has shown the existence
of left handed coiling in DNA, commonly referred as Z-DNA or Z form. The sugar phosphate
backbone of this DNA follows a zigzag path containing 12 base pairs in one turn and is
assumed to play role in gene regulation. Other than these (A, B, & Z form), there are other
conformations of DNA which are found to occur in controlled or experimental condition.

FEATURES A B Z
Helical sense Right handed Right handed Left handed
Diameter 25.5 Å 20 Å 18.4 Å
Base-pairs per helical turn (n) 10.7 10.4 12
Helical twist (or Rotation) per bp 33.6º 34.3º 60º / 2 bp
(360/n)
Rise per bp 2.3 Å 3.3 Å 3.8 Å
Base tilt to helix axis 20º 6º 7º
Major groove Narrow and deep Wide and deep Flat
Minor groove Wide and shallow Narrow and deep Narrow and deep
Helix pitch 24.6 Å 34.3 Å 45.6 Å
Mean propeller twist +18º +16º 0º
Glycosyl-bond conformation anti anti anti at C, syn at G
Sugar pucker C3’-endo form C2’-endo form C2’-endo for
pyrimidine and
C3’-endo for
purines.
Table: Features of A, B and Z type of DNA

Viral DNA
The central core or genome of a virus may only have a single type of nucleic acid. They may
either have ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) and are known as RNA
virus or DNA virus respectively. Once the DNA is injected into the host cell, it will enter the
nucleus of the host cell, where the viral DNA can integrate with the host cell genome. The

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viral DNA utilises the host cell’s polymerase enzyme to replicate the viral DNA and produces
new viruses. Both DNA and RNA are never found together in a virus. Nucleic acid in virus
can either single stranded or double stranded, with a circular, linear or segmented
arrangement. DNA virus can be divided into two groups; i.e. double stranded DNA (dsDNA)
viruses, and single stranded DNA (ssDNA) viruses. Double stranded DNA (dsDNA)
viruses are considerably complex and its size varies from 5 kbp in polyomaviruses to 130–
365 kbp in poxviruses. Viruses with genome more than 300 kbp are known as ‘giant virus’,
although giant viruses had been known for a few years. Mimivirus is the largest virus
ever discovered that infect amoebae. In dsDNA, during transcription a preinitiation complex
binds with the positive strand and RNA polymerase uses negative strand as template. dsDNA
viruses use different mechanism, i.e. Bidirectional replication mechanism, Rolling circle
mechanism, Strand displacement mechanism and Replicative transposition mechanism, to
replicate viral genome in a host cell. Single stranded DNA (ssDNA) viruses are
comparatively small and simple DNA viruses and its size varies from 2-6 kb in length. These
viruses infect diverse hosts from all three domain of life. ssDNA viruses depends on the host
cell for much of their replication machinery. For transcription, ssDNA is made into dsDNA
by DNA polymerase after entering a host cell, mRNA is then synthesized in same manner.
The DNA in these viruses can be either circular or linear, the former ones are replicated by
rolling circle replication (RCR) mechanism with the help of endonucleases, while the linear
one ‘hairpin’ structures at the end of DNA play a part in DNA replication.

Bacterial DNA
The main distinguishing feature between prokaryotic cell and eukaryotic cell is that in
prokaryotic cell nuclear membrane is absent. The DNA present in bacteria is of two types-
Genomic DNA and Plasmids. Similar to eukaryotic DNA, bacterial DNA performs the
function of encoding of all of the information needed to program the cell's activities including
reproduction, metabolism, etc. Bacterial cells lacks true nucleus or a nuclear membrane and
do not possess the complex chromosome of the kind as present in eukaryotes. Instead, the
genes are encoded within DNA, which is packed and localised in dense zone of cytoplasm of
the cell. In most of the bacteria the DNA is a single circular molecule, called the bacterial
chromosome. The chromosome, along with several proteins and RNA molecules, forms an
irregularly shaped structure known as the nucleoid. Most of the bacteria have a haploid
genome. Usually, the bacterial genome is represented by long, single circular DNA attached
to a cell membrane, sometimes a closed loop of DNA is also present. Not all bacteria have a
single circular chromosome: some bacteria have multiple circular chromosomes. Sometimes
linear chromosomes are also reported in some bacterial cell. A bacterial cell contains
approximately 0.5–20 fg DNA. When the bacterial cells are immersed in a medium having
high refractive index then with the application of phase-contrast microscopy nucleoid region
can be observed. Nuclear bodies were first observed with the help of Giemsa and Feulgen
stains using light microscope.

Plasmid DNA
Many bacterial cells, in addition to nucleoid, possess small, independently replicating circles
of double stranded DNA called plasmids. They are best thought of as an excised portion of

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the bacterial chromosome. Normally plasmid DNA occurs independently in cytoplasm of

cells or it may also be found in associated with main chromosomal DNA, called episome.
Plasmids contain only a few genes (apart from main genome) that are usually not vital for the
cell’s survival but they carry genes that encodes different proteins which aid the host cell to
perform specific functions viz. drug resistance, mating ability, toxin production, etc.

MITOCHONDRIAL AND CHLOROPLAST DNA

Mitochondrial DNA
The mitochondria are a highly specialized organelle present almost in all the cells. The
functions of mitochondria are well conserved across species. They generally encode proteins
for oxidative phosphorylation (energy production), in addition to this, mitochondria are
involved in many other activities, it plays a vital role in calcium signalling, it also play role in
biosynthesis of heme, steroid, amino acids and regulates cellular metabolism, and apoptosis.
Mitochondria, independent from their nucleus, they possess their own genetic material and
the machinery to manufacture RNA and proteins.

The genetic material in mitochondria is small, circular DNA and known as mtDNA. In case
of plants, mtDNA exists as a collection of linear DNA with combinations of small circular
and branched molecules. A study performed by Lo et al. (2011) went into extensive detail
concerning the structure of DNA in mung bean mitochondrial nucleoids. They observed plant
mtDNA as supercoiled molecules, sub-genomic open circles of variable size, linear
molecules, and other highly complex structures. mtDNA encodes for small number of
polypeptides that are tightly integrated into the inner mitochondrial membrane along with the
other polypeptides, forming a DNA-protein complexes in the mitochondrial matrix. In
comparison to nuclear genome, the mitochondrial genome is relatively short with ca. 16,500
base pairs and 37 genes (28 on H-strand and 9 on L-strand). Plant mitochondrial genomes are
relatively larger and more complex, than of animals. The plant mitochondrial genomes range
between 200-2,000 kbp. Irrespective of the genome size of plant mitochondria, it codes for
relatively few genes. With the increase in size of mitochondrial genome the genes does not
increases. The increased size of mitochondrial DNA is due to the presence of repeated
sequences, AT-rich non-coding regions, and large introns and non-coding sequences.

Both mitochondrial and nuclear DNA works in coordination to regulate energy production.
mtDNA differs transcriptionally from the nuclear DNA of any eukaryotic cell. Nuclear DNA
produces messenger RNA that is monocistronic. On the other hand, the mitochondrial DNA
is polycistronic. According to Lynn Margulis, mitochondrial genome has been derived from a
eubacterial ancestor through endosymbiosis. It means that genome of plant mtDNA is a
remnant of its prokaryotic symbiotic ancestor. It is considered that this genome must have
been much larger earlier but it evolved rapidly in structure because of its extensive
recombination activities. Because of the size and complexity of plant mitochondrial genomes,
the exact mechanism for plant mtDNA replication remains unclear. Multiple hypothesis viz.
recombination-dependent replication, rolling circle mechanism, bidirectional replication
method, etc. has been given by different researchers to explain the mtDNA replication.

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Chloroplast DNA
The chloroplast is a well-known green coloured plastid found in all the photosynthetic cells
of plant. It comprises of green-coloured pigments called chlorophyll and is a membrane
bound organelle that converts light energy into chemical energy and provide plants with
foods and energy in the form of sugar or starch by the process of photosynthesis. Apart from
nuclear DNA, chloroplast has its own DNA, involved in the preservation, replication, and
expression, known as chloroplast DNA (cpDNA). The DNA in chloroplast is maternally
inherited, circular and is localized in specific regions of the plastid and sometimes is called
plastidome. Chloroplast genome characteristically exhibits the presence of two identical
inverted repeats in the DNA sequence. The first existence of chloroplast DNA was first
suggested in 1951. Later, in 1962, Ris and Plaut cytologically provided the first convincing
evidence that chloroplasts contain DNA.

cpDNA codes for proteins and RNAs that is crucial and plays a significant role in metabolic
processes. It encodes for rRNA, tRNA, larger subunit of RUBISCO and many proteins
participating in photosynthesis and other essential processes like gene expression and
electron transport. Although vast majority of the proteins of chloroplast are encoded by
nuclear DNA and are produced in cytosol of plant and transported to organelle by specialized
transport machinery. In comparison to mitochondria, the amount of DNA per chloroplast is
much greater. mtDNA and plastid DNA together amounts for about one-third of the nuclear
DNA. Replication of chloroplast DNA is independent of nuclear DNA replication. The
chloroplast genome ranges between 70-200 kbp and encodes for approximately 120 genes,
having the contour length of 30-60 mm and mass of about 80-130 mDa. A single mesophyll
chloroplast is able contain up to 300 chromosomes organized into nucleoids. These are
complex structures each consisting of 10-20 copies of the plastid genome along with RNA
and various proteins (Krupinska et al, 2013). The first complete study of chloroplast genome
was done in tobacco (Nicotiana tabacum) in 1986. Chloroplast genome comprises of 04
genes for rRNA, 20 for ribosomal proteins, 30 for tRNAs, many of the photosynthetic
proteins, 6-9 genes for ATPase, and chloroplast RNA polymerase.

It has been considered that chloroplasts are originated from photosynthetic prokaryotes that
became part of a plant cell by endosymbiosis. In contrast to the evolution of the nuclear DNA
in the plant cell, cpDNA has evolved at a more conservative pace and exhibits a more
interpretable evolutionary pattern. Over time, there appears to have been some genetic
exchange between the nuclear and chloroplast genomes. Both morphological and molecular
studies on chloroplast can provide a clearer understanding of its evolutionary processes.

S. No. Properties Mitochondrial DNA Chloroplast DNA

1 Present in all eukaryotes plant cell
2 Organelle mitochondria of the cell chloroplast of the cell
3 Shape circular and linear circular
4 DNA type double stranded. double stranded.
5 Abbreviation mtDNA cpDNA
6 No. of genes 37 200
7 Existence as multiple copies as multiple copies
8 Distribution randomly distributed to daughter randomly distributed to daughter cells

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cells
9 No. of base pairs 16.5 kbp in humans 70-200 kbp
200-2000kbp in plants
10 Histone proteins absent absent
11 Introns absent absent
12 Functions genes in the mitochondrial DNA chloroplast DNA participate in
take part in oxidative photosynthesis in plants.
phosphorylation for the
production of ATPs and in the
synthesis of proteins.
Table: Properties of Mitochondrial DNA and Chloroplast DNA

1.7 RNA SYNTHESIS AND ITS TYPES

RIBONUCLEIC ACIDS (RNA)
After the discovery of double helical model of the DNA by Watson and Crick in 1953,
scientists turned to RNA structure and said that it is usually a single stranded molecule
consisting of a long chain of nucleotide units attached by phosphodiester bond. Each unit of
RNA nucleotide consists of a nitrogenous base, a ribose sugar, and a molecule of phosphate.
Similar to DNA, RNA has four nitrogenous bases, but unlike DNA, it has uracil (U) instead
of thymine (T). Thus, the four RNA nucleotides are adenine (A), cytosine (C), guanine (G),
and uracil (U). During complementary base pairing with DNA, G pairs with C, T in DNA
pairs with A in RNA, A in DNA pairs with U in RNA. While during RNA-RNA base pairing,
G and C pairs with each other and A and U pairs together.

RNA Synthesis
Francis Crick explains the flow of genetic information in biological system and proposed
Central Dogma of Molecular Biology. He explained the directional flow of genetic
information from DNA to RNA to protein. The process of by which molecules of RNA is
synthesized from the genetic information encoded on DNA template with the help of enzyme
RNA polymerases called transcription. DNA segment, that are transcribed into RNA
molecule and synthesize protein, produces mRNA (also known as coding RNA), whereas
other segments that are copied into RNA molecule forms non coding RNAs (ncRNAs). RNA
polymerases use nucleotide triphosphate (ATP, UTP, GTP and CTP) and a template of DNA.
During the process of RNA synthesis in prokaryotes one type of RNA polymerase is used
while in eukaryotes 3 types of RNA polymerases are used. RNA polymerase I transcribes
ribosomal RNA (rRNA), pol II transcribes mRNA and pol III tRNA.

During transcription three different types of RNAs are synthesized:

i. mRNA (or Messenger RNA): carries message for protein synthesis.
ii. tRNA (or transfer RNA): carries amino acid during protein synthesis.
iii. rRNA (or ribosomal RNA): forms the part of ribosomes.

Types of RNA
RNA is classified on the basis of their nature and function. On the basis of nature, RNA is of
two types, namely genetic RNA and non-genetic RNA.

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Genetic RNA: In some organisms viz. some viruses, RNA act as hereditary material. It
carries genetic information from parent to offspring. It has self replicatory property. It can be
double stranded or single stranded.

Non-genetic RNA: On the basis of functioning, RNA is classified into three types, namely
mRNA, tRNA and rRNA. Non genetic RNA does not serve as hereditary material but is the
one that is transcribed by DNA.

mRNA (Messenger RNA): The name messenger RNA (mRNA) was proposed by Francois
Jacob and Jacques Monod in 1961, it carries the genetic information contained in DNA which
control the cellular activities. It is synthesized in the nucleus of cell from DNA and then
carried out of the cell to facilitate protein translation. The cell does not contain large
quantities of mRNA, accounting for only 5% - 10% of the total RNA in the cell. This is
because mRNA constantly undergoes breakdown into its constituent ribonucleotides by
ribonucleases, unlike rRNA and tRNA. The main function of mRNA is to carry genetic codes
copied from DNA in nucleus in the form of triplets, known as codons, during the process of
transcription to the site of protein synthesis i.e. ribosome (present in cytoplasm).

The structure of mRNA

The molecule of messenger RNA (mRNA) consists of single strand having A, G, U and C
nitrogenous bases. The main purpose of mRNA is that it functions as a template for protein
synthesis, it carries genetic information from DNA (in nucleus) to a ribosome (in cytoplasm)
and assemble amino acids properly. Each amino acid in a protein is coded by the sequence of
three nucleotides which together form a unit of genetic code is called codons. The structure of
mRNA molecule comprises of following segments:

5’ Cap : This region has a methylated structure at 5’ end and is associated with binding of m-
RNA molecules to ribosomes.

Non-coding region: The 5’ cap is then followed by a non coding region with length of 10-
100 nucleotides. This region is rich in A and U residues. As per its name it does not code for
any protein.

Initiation codon: This is the triplet which marks the beginning of protein synthesis. It is
AUG and codes for Methionine.

Coding region: This region is between initiation codon and termination codon and contains
the sets of triplet codon which codes for different types of amino acids.

Termination codon: These are the codon that signals the termination of translation. These
codons are also known as nonsense or stop codons. For these codons no molecule of tRNA
exist. There are three nonsense codons are called UAG (Amber), UAA (Ochre) and UGA
(Opal).

Another non coding region: This region consists of 50-150 nucleotides with a specific
sequence AAUAAA.

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3’Poly-A tail: This is the last segment of mRNA consisting of 200-250 nucleotides. It consist
of large number of A residues.

rRNA (Ribosomal RNA): Ribosomal RNA or rRNA, as its name suggest, is present in
ribosomes and constitutes about 80% of total cellular RNA. Among all the different types of
RNA, rRNA is most stable and insoluble. It is a single stranded which might gets twisted and
forms complementary base pair within the strand. It unfolds on heating and got refolded on
cooling. The ribosomes are composed of two major nucleoprotein subunits, the eukaryotic
80S ribosome has 60S and 40S subunit whereas the prokaryotic 70S ribosome has 50S and
30S subunits. Each ribosomal subunit comprises of one or more rRNA and a variety of
proteins. In eukaryotes 40S subunit contains of 18S rRNA and 50S subunit contains 28S
rRNAs, 5.8S rRNAs and 5S rRNAs. In prokaryotes 30S subunit contains of 16S rRNA and
50S subunit contains 5S rRNAs and 23S rRNAs. Small subunit of ribosome reads the RNA
and the larger one joins the amino acid to synthesize protein.

The functions of rRNA molecules are little known. It is the part of the protein-synthesizing
organelle. The primary function of rRNA is in protein synthesis ensuring the proper
alignment of the mRNA and the ribosomes. rRNAs combine with proteins in the cytoplasm
and has the enzymes needed for the process. rRNA also act as enzymes, known as ribozymes,
having role introns splicing.

tRNA (Transfer RNA): Transfer RNA (tRNA), also known as soluble RNA, is the smallest
RNA among the 3 types of RNA with 71-80 nucleotides. Its molecular weight ranges from
25000 to 30000. Apart from usual nitrogenous bases tRNA contains unusual bases with some
modifications, viz., methyl adenine, methyl cytosine, methylguanine, pseudouracil and
others, which make it unique from other RNAs. Each tRNA molecule binds with specefic
amino acid and transfers it to the site of protein synthesis and recognizes the codons of the
mRNA. Hence, tRNA molecule acts as an adapter between mRNA and amino acid. Earlier it
was considered that there are 20 tRNA molecule for 20 aminoacid i.e. one tRNA for each
amino acid. Later it was reported that at least two types of tRNA exist for one amino acid.

Structure of tRNA
The primary structure of tRNA was first given by Holley et al. (1965) in yeast and presented
the clover leaf model. It is stabilized by strong hydrogen bonds between the nucleotides.
Clover leaf model of tRNA consists of 5 arms made up of single polynucleotide chain and
three structural loops that are formed by hydrogen bonding. The 3’ end serves as the site of
amino acid attachment and three bases 5’-CCA-3’ are added to the 3’ end of every tRNA
molecule.

Five arms or parts of cloverleaf model are as follows:

i. Acceptor arm: Amino acid attaches to this region of tRNA, hence also named as amino
acid carrier arm. It is 3’ terminal end of acceptor arm that attaches amino acid. Base pairing
in 3’ terminal end is absent. 3’ end of every tRNA molecule is capped by the nucleotide
sequence CCA-OH (5’-3’).

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ii. Anticodon arm: This arm of tRNA, consisting of seven unpaired bases out of these three
nucleotide bases which is complementary to triplet codon of mRNA forms the anticodon, is
responsible of recognising the codon. mRNA attaches to this site of tRNA and transport
amino acid to protein synthesis site.

iii. D-arm or D-loop or DHU loop: Due to the presence of an unusual or modified
pyrimidine base dihydrouridine it is named so. This arm helps in attachment of amino-acyl
synthetase.

iv. TΨC arm: This region contains thymidine, pseudouridine and cytidine residues hence
named TΨC arm or T loop. This region recognises and determines the ribosomal site where
tRNA molecule has to attach during protein synthesis.

v. Variable arm: Also known as extra arm, this is the most variable region in tRNA
molecule. Nucleotides composition is variable and is lacking entirely in some tRNA
molecule. On the basis of variability it is classified into two classes, viz. Class I tRNAs (The
dominant form, with 3-5 base pairs long) and Class II tRNAs: (with 13-20 base pair long
arm)

Function of tRNA: tRNAs are an essential component of translation. The main function of
transfer RNA is to bring or transfer amino acids to the ribosome that corresponds to each
triplet codon of mRNA. The amino acids are then joined together and processed to form
polypeptide chains and proteins.

S. No. Features mRNA rRNA tRNA

1. Constitutes total 5-10 % 80% 10-15%
percent of total
cellular RNA
2. Sedimentation 8S 28S, 18S, 5.8S, 3.8S
cofficient 5S, 23S, 16S, 5S
3. Molecular 50000 23S: 1.1 X 105 25000-30000
weight 30S: .55 X 106
4. Unusual bases less less high
5. Site of synthesis Periphery of nucleolus Centre of Outside nucleolus
nucleolus
6. Base-DNA Bases of mRNA are Forms from a Forms from a
relation complementary to small part of very small part of very
DNA bases less DNA less DNA
7. Function carry genetic codes ensures the proper transfer amino
copied from DNA in alignment of the acids to the
nucleus in the form of mRNA and the ribosome that
triplets, known as ribosomes during corresponds to
codons protein synthesis each triplet codon
of mRNA

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8. Shape linear spherical Clover leaf model

9. Presence of Carries codons codons or Carries anticodons
codons or anticodons are
anticodon absent
Table: A comparative study of different types of RNA

1.8 POST TRANSCRIPTIONAL MODIFICATION

During the process of transcription (or RNA synthesis) RNA is transcribed but before the
translation it must be processed to generate a mature and functional RNA. If the RNA is not
processed then no protein synthesis will take place. This process of generating a mature
functional RNA is called Post-Transcriptional Modification (PTM). It includes
phosphodiester bond cleavage, addition or alterations of existing bases or sugars, specific
cleavage or modification of nucleotide or introns splicing. The primary transcript formed by
RNA polymerase enzyme is called as pre RNA. In prokaryotes pre mRNA molecule undergo
little or no modification, whereas in eukaryotic organisms it is significantly modified. In both
prokaryotes and eukaryotes pre rRNA and pre tRNA undergo modification to become rRNA
and tRNA.

Types of post transcriptional modifications

In post transcriptional modifications pre RNA, before being transported to cytoplasm,
involves 5’ end capping, 3’-OH polyadenylation and RNA splicing.

5’ Capping: It is an enzyme catalysed process, in this 7-methylguanosine residue is added to

5’ end of nascent RNA. When the mRNA transcript is 20-25 nucleotides in length, RNA
triphosphate removes one of the three phosphates at 5’ end. Guanosine Mono-Phosphate is
added at 5’ diphosphate forming Guanosine 5-5 triphosphate. Finally, enzyme
methyltransferase methylates the newly attached guanine. The main function of 5’ end
capping is that it protects mRNA from degradation, it transports the mRNA to cytoplasm
from nucleus and it also aid mRNA in binding with ribosomes.

3’-OH, polyadenylation: A poly A tail (consisting of about 50-250 nucleotides) is added to

3’ end of nascent mRNA transcript without the involvement of DNA template. This process
is catalysed by template independent RNA polymerase enzyme called as poly (A)
polymerase. Polyadenylation process took place in two steps: (i.) Cleavage of nascent RNA
at internal site (not at extreme 3’ end) creates a new 3’ end, (ii.) Addition of poly (A) tail with
the help of polymerase and binding protein. The functions of 3’-OH Polyadenylation are that
it export mature mRNA from nucleus, it affect the stability of mRNA and serves as
recognition signal for the ribosomes.

RNA Splicing: In eukaryotic organism interrupted coding regions are present. In this process
the non-coding sequences (i.e. introns), which are present in-between the nascent mRNA
transcript, are removed and protein-coding sequences (i.e. exons) are ligated. Hence, it is also
known as Introns splicing. The non-coding sequences of transcript are removed by two ways:

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(i.) Self splicing or auto splicing: This occurs in some rare introns that help in the cleavage of
phosphodiester bond and formation without the help of spliceosomes. In this process RNA
itself act as enzyme to remove the non-coding regions from the mRNA transcript.
(ii.) Alternative splicing: In this process exons from the same gene are joined in varying
combinations, by this several different functional mRNA transcripts are produced.

1.9 GENETIC CODE

Twenty different types of amino acids involved in the synthesis of polypeptides are present in
nature. Single and double base coding will not cover all the 20 amino acids. A single genetic
code involving each nucleotide, coding for single amino acid, can code only for one
polypeptide with four amino acids, while the two bases, codes for 16 amino acids in the
synthesis of protein. Crick suggested that each amino acid is a specified by a triplet of
nucleotides i.e. codon. Marshall Nirenberg and Heinrich Mathaei used the tailor made
artificial triplets of RNA in cells free systems to experimentally prove that a sequence of
three nucleotide bases codes necessary for a single amino acid. The three nucleotide bases in
mRNA constitute the genetic code or codon. This codon act as a code word for the synthesis
of protein. The genetic code is regarded as a dictionary of nucleotide bases, viz. A, G, C and
U, that determines the sequence of amino acid and proteins.

Characteristics of Genetic Code

The code is degenerate: Particular amino acid can be specified by more than one triplet code
on except for material and tryptophan
Code is triplet: Code is triplet of three nitrogenous bases constitute a triplet call this codes
on which specify a particular amino acid on a polypeptide chain
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Code is unambiguous: Each triplet code on specifies only one single amino acid
Code is non-overlapping: Change in single nitrogenous base could affect more than one
codon Effecting more than one amino acid acid
Code is commaless: The second Amino acid is automatically coded coded by the next triplet.
There is no space for punctuation in between every codon

1.10 PROTEIN SORTING OR PROTEIN SORTING

Majority of the proteins are synthesized in 80S ribosomes present in the cytosol of the cell,
whereas the few proteins are synthesized on 70S ribosomes of mitochondria and chloroplast.
In both eukaryotic and prokaryotic cell each of the newly synthesized proteins are sorted with
the help of molecular labels (specific amino acids) and then transported to the specific
location such as inner space of an organelle, intracellular membranes, plasma membrane, etc.
of cell in which it functions. This process of transporting newly translated protein to a
particular destination is known as protein targeting or protein sorting. Protein targeting or
protein sorting is the biological mechanism by which proteins are identified and correctly
transported to their appropriate positions in the cell or outside of it.

Targeting signals or Sorting signals

Synthesized polypeptide chains contain information that facilitates the cellular transport
machinery to accurately target the protein from cytosol to a specific organelle (for eg.
nucleus, endoplasmic reticulum, mitochondria, peroxisomes) inside or outside the cell. These
pieces of information are known as targeting signals or sorting signals. Without targeting
signals proteins cannot be transported to their specific destination. They remain permanently
in the cytosol.

The delivery of newly synthesized proteins is done mainly by two different types of
translocation processes:
(i) Co-translational transport
(ii) Post-translational transport

Co-translational Transport: This type of transport occurs in the proteins that are being
translated in the ribosomes of RER. This protein transport occurs along with the process of
translation. During the process of translation, proteins (along with the ribosomes) are initially
brought to the endoplasmic reticulum. From ER, proteins enter endomembrane system i.e. it
is transported to golgi apparatus in membrane vesicles. Here protein may undergo some
modifications and then go to final destination. From the Golgi apparatus, protein may be
transported to plasma membrane, lysosomes or plasma membrane. The proteins transported
in this process may be soluble proteins or secretory proteins or integral proteins.

Signal sequence: A hydrophobic chain of 6-12 hydrophobic amino acids present on N

terminal of nascent polypeptide chain.
Signal recognition protein (SRP): A ribonucleic protein, formed of 1 RNA and 6 proteins,
recognize and binds with the signal sequence, smaller subunit of ribosome and SRP receptor
on ER.

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Translocon: A channel, made up of 3 subunits, is present on ER membrane which prevents

the leakage of calcium from ER lumen to cytoplasm.

Proteins of SRP will recognize and binds with the signal sequence of protein and smaller
subunit of ribosomes forming a protein-ribosome-SRP complex. This blocks the N terminal
of polypeptide chain and smaller subunit of ribosome, therefore preventing the addition of
amino acids and the translation is paused. The complex is transported to SRP receptor in ER.
SRP of complex binds with SRP receptor of ER with GTP and larger subunit (with N
terminal) attaches to translocon. GTP will be hydrolysed and translocon will open. SRP will
be released from the complex, nascent protein will enter the translocon channel and
translation will restart. Signal peptidase enzyme present in ER will cut the signal sequence.
When translation is completed Ribosomal Releasing Factor (RRF) will remove the attached
ribosome. The newly formed protein is covered by Chaperone protein which aids in its
folding. Folded protein is then transferred to golgi apparatus and then to targeted organelles.

Post-translational transport: Some of the proteins are translated on free ribosomes i.e.
those present in cytosol. In prokaryotes a closed membrane protein (Sec Y) is present on
plasma membrane. A enzyme Sec A (ATPase enzyme), continuously breaks ATP to ADP,
will bind SecY and facilitate the entry of protein inside the extracellular fluid. In eukaryotes
chaperon proteins binds with the translated protein. The N terminal of signal sequence of the
complex interacts with the translocon (Sec 61) of ER. The translocon opens and facilitate the
entry of protein inside the lumen. Signal peptidase, enzyme present inside the lumen, will
cleave the signal sequences present in the protein. A BiP (Binding immunoglobulin protein)
protein present in the lumen binds with the protein (without N terminus), hydrolyses ATP to
ADP with the help of Sec 63 complex. Energy due to ATP hydrolysis helps in pulling of
protein little by little. This hydrolysis and pulling will continuously take place until whole
protein is pulled inside the lumen. BiP is removed from the completely pulled protein is
released into the ER lumen.

1.11 SUMMARY
DNA or RNA is the principal genetic materials made up of linear polymer of nucleotides.
Each nucleotide comprise of three parts viz. phosphate group, sugar (either ribose or
deoxyribose) and heterocyclic nitrogenous bases (related to either purine or pyrimidine).
Based on the x-ray diffraction image and Chargaff’s rule Watson and Crick successfully
deduced a three dimensional structure of DNA. They reported that DNA is formed of two
complementary strands of polynucleotides twisted around each other in the form of double
helix running in opposite direction. There are approximately 10 bases per turn in DNA. Two
adjacent bases are separated by the distance 3.4 Å, the diameter of the helix is 2 nm. Two
grooves (major & minor) of different width are formed due to asymmetrical spacing of the
sugar-phosphate backbones between the adjacent turns. There are three main types of DNA,
A, B and Z type. Experiments conducted by Griffith in 1929, Hershey and Chase in 1952
and Meselson and Stahl in 1958 demonstrated that DNA is the genetic material, rather than
protein.

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In some living organism, other than DNA, RNA is genetic material. Similar to DNA, RNA
has four nitrogenous bases, but unlike DNA, it has uracil (U) instead of thymine (T) and the
sugar present in RNA is ribose. RNA is synthesized from the genetic information encoded on
DNA template with the help of enzyme RNA polymerases called transcription. There are
three different types of RNAs mRNA (or Messenger RNA), tRNA (or transfer RNA) and
rRNA (or ribosomal RNA). The mature RNA is later translated to synthesize the polypeptide
chain encoded by the original gene. But before the process translation, the RNA must be
processed to generate a mature and functional RNA. Twenty different types of amino acids
involved in the synthesis of polypeptides are present in nature. Each amino acid is a specified
by a triplet of nucleotides i.e. codon.

1.12 BIBLIOGRAPHY
Y. S. Lo, L. J. Hsiao, N. Cheng, A. Litvinchuk and H. Dai: Characterization of the structure
and DNA complexity of mung bean mitochondrial nucleoids. Mol Cells, 31(3), 217-24
(2011).

1.13 SUGGESTED READINGS

 A Textbook of Biotechnology – R. C. Dubey
 Lehninger Principles of Biochemistry- Nelson and Cox
 Elements of Biotechnology – P. K Gupta
 Biotechnology Fundamentals and Applications – S. S. Purohit

1.14 TERMINAL QUESTIONS

Q1. Explain physical and chemical structure of DNA. Discuss Watson and Crick model of
DNA.
Q2. What are different types of DNA? Discuss.
Q3. Describe about different experimental approaches which established DNA to be the
universal genetic material?
Q4. What are different types of RNA? Discuss.
Q5. Explain Post-transcriptional modification of RNA.
Q6. What do you understand by Protein sorting?
Q7. What is Genetic Code?

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UNIT-3- PROKARYOTIC AND EUKARYOTIC REGULATION

OF GENE EXPRESSION

3.1-Objectives
3.2-Introduction
3.3-Prokaryotic and Eukaryotic regulation of gene expression
3.3.1-Prokaryotic regulation of gene expression
3.3.2-Eukaryotic regulation of gene expression
3.4-Summary
3.5-Glossary
3.6-Self Assessment Question
3.7-References
3.8-Suggested Readings
3.9-Terminal Questions

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3.1 OBJECTIVE

After reading this unit you will be able to:

This topic about control of gene expression focuses on various aspects of gene regulation
and the mechanisms.

1. Know about the basic facts and mechanisms of gene expression.

2. Study this gene regulation or gene expression by considering suitable examples.
3. Understand how genes regulating the expression and the role of RNAs and its various
forms.
4. Understand the difference between prokaryotic and eukaryotic gene regulation.

3.2 INTRODUCTION

As we know the central dogma, a major theme of molecular biology states that the genetic
information flows from DNA to RNA and RNA to proteins. This provides a molecular basis
for the connection between genotype and phenotype but failed to answer that how this flow
of information along the molecular pathway regulated.

Now in this chapter we consider one of the most fundamental questions: How is genetic
expression regulated? Because it’s clear that not all genes are expressed at all times in all
conditions or situation, their expression shows variations depending upon different condition
in a unit time. The basics cellular function of an organism is influenced by cellular
environment and the adaptation to specific environments is achieved by regulating the
expression of genes that encode the enzymes and proteins needed for survival in a particular
environment.

The level of gene expression may differ from one cell type to the next or according to stage in
the cell cycle. The expression of genes varies according to the need and specialization of the
cell. For examples an animal contains numerous cells of define functions and with the minor
exceptions, all having same genetic composition, but differ in function only due to the
expression of genes. In multicellular plants and animals, gene expression is under elaborate
control whereas in the single – celled bacterium it can be as simple as switching on and off of
gene to make the enzymes needed to digest whatever food sources are available. For
example, the β cells of the pancreas makes the insulin and the α cells of the pancreas make
glucagon, in another example the genes for hemoglobin are expressed at high levels only in
precursors of the red blood cells. The difference between all these cells is because of the
precise control of gene expression. Factors that may influence gene expression include

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nutrients, temperature, light, toxins, metals, chemicals, and signals from other cells.
Malfunctions in the regulation of gene expression can cause various human disorders and
diseases.

The gene expression system or regulatory systems of prokaryotes and eukaryotes are
somewhat different from each other. The process is essential for prokaryotes and eukaryotes
as it increases the versatility and adaptability of an organism by allowing the cell to express
protein when needed. In both, prokaryote and eukaryotes gene expression can be regulated at
a number of different points. In prokaryotes, they generally adapted to free-living, unicellular
and simple structure symmetry of life, in which they grow and divide indefinitely as long as
environmental conditions are suitable and the supply of nutrients is adequate. Their systems
are provided to utilize the growth condition or unit conditions in unit time to attain the
maximum growth rate. While the most eukaryotes are not only adapted to utilize the growth
condition or unit conditions but the progeny cells must also undergo considerable changes in
morphology and biochemistry and then each maintain its altered state. In eukaryotes the first,
regulation can be through the alteration of DNA or chromatin structure. A second point at
which a gene can be regulated is at the level of transcription. A third potential point of gene
regulation is mRNA processing. A fourth point for the control of gene expression is the
regulation of RNA stability. A fifth point of gene regulation is at the level of translation, a
complex process requiring a large number of enzymes, protein factors, and RNA molecules.
Finally, many proteins are modified after translation and these modifications affect whether
the proteins become active; so genes can be regulated through processes that affect
posttranslational modification.

Further, on the basis of assigned function some structural genes, particularly those that
encode essential cellular functions, are expressed continually and are said to be constitutive
and therefore not regulated by any regulatory gene or their products. While few DNA
sequences that are not transcribed at all but still play a role in regulating genes and other
DNA sequences. These regulatory elements affect the expression of sequences to which they
are physically linked. In this way, prokaryotes and eukaryotes use regulatory genes to control
the expression of many of their structural genes. The regulation of gene expression can be
through processes that stimulate gene expression, termed positive control, or through
processes that inhibit gene expression, termed negative control.

In this chapter, we discuss and explore regulation of gene expression in prokaryotes and
eukaryotes. Before considering gene regulation in both bacteria and eukaryotes, we must
distinguish between the DNA sequences that are transcribed and the DNA sequences that
regulate the expression of other sequences. According to one of the basic concept, genes
include DNA sequences that encode proteins and as well as sequences that encode rRNA,
tRNA, snRNA, and other types of RNA required to stabilize the integrity of cell and
organism. On the basis of allotted function within the cells group of genes tends to work
together to foster the functioning of the cells. Group of genes or structural genes encode

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proteins that are used in metabolism or biosynthesis or that play a structural role in the cell.
Similarly regulatory genes are genes whose products, either RNA or proteins interact with
other DNA sequences and affect the process of transcription or translation.

3.3 CONTROL OF GENE EXPRESSION IN PROKARYOTES

AND EUKARYOTES

3.3.1 Gene regulation in prokaryotes

The idea that microorganism regulated their gene product to different conditions is not new.
Two French microbiologists, Jacob and Monad (1961) found that the genetic material
possesses group of regulatory gene units called operons in prokaryotes for which they
received Nobel Prize in 1965. Jacob and Monad proposed that the transcription of a set of
contiguous structural genes is regulated by two controlling elements. The regulation of gene
expression has been extensively studied in many prokaryotes, particularly in E. coli. In
bacteria the genes that have related functions are clustered and under the control of a single
promoter. These genes are often transcribed together into a single mRNA. A group of
bacterial structural genes that are transcribed together is called an operon. In prokaryotic
cells, there are three types of regulatory molecules that can affect the expression of operons:
repressors, activators, and inducers.

Repressors and activators are proteins produced in the cell. Both repressors and activators
regulate gene expression by binding to specific DNA sites adjacent to the genes they control.
In general, activators bind to the promoter site, while repressors bind to operator regions.
Repressors prevent transcription of a gene in response to an external stimulus, whereas
activators increase the transcription of a gene in response to an external stimulus. Geneticists
have developed that idea, that efficient genetic mechanisms have evolved in these organisms
to turn transcription of specific genes on and off, depending on the environmental conditions
and cell’s metabolic need for the respective gene products.

A general structure of an Operon

A typical operon Figure 3.1. In general organization of Operon, a set of structural genes lies
at the end of the operon, these structural genes are transcribed into a single mRNA, which
than translated to produce enzyme and then translated enzyme produce product of interest.
For example, structural genes (as gene a, gene b, and gene c) at one end of the operon These
structural genes are transcribed into a single mRNA, which than translated to produce
specific enzymes A, B, and C and these enzymes further carried out a series of biochemical
reactions that convert precursor molecule O into product P. The transcription of structural
genes a, b, and c is under the control of a promoter, which lies upstream of the first structural

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gene. RNA polymerase binds to the promoter and then moves downstream, transcribing the
structural genes.

Control of operons

In general the above said mechanism is controlled by a regulator gene. A regulator gene has
its own promoter and is transcribed into a short mRNA, which is translated into a small
protein. This regulator protein can bind to a region of the operon called the operator and
affect whether transcription can take place. There are two types of transcriptional control:

(i) Negative control, in which a regulatory protein is a repressor, binding to DNA and
inhibiting transcription.
(ii) Positive control, in which a regulatory protein is an activator, stimulating
transcription.

Operons can also be either inducible or repressible.

(i) Inducible operons are those in which transcription is normally off and something
must happen to induce the transcription or to turn it on.
(ii) Repressible operons are those in which transcription is normally on and something
must happen to repress the transcription or turn it off.

Figure 3.1: General overview of Operon (I=regulatory region, P=promoter region and O=
operator region)

Negative inducible operons: In a negative inducible operon, the regulator gene encodes an
active repressor that readily binds to the operator. Because the operator site overlaps the
promoter site, the binding of this protein to the operator physically blocks the binding of
RNA polymerase to the promoter and prevents transcription. For transcription to take place,
something must happen to prevent the binding of the repressor at the operator site. This type
of system is said to be inducible, because transcription is normally off (inhibited) and must be
turned on (induced). Transcription is turned on when a small molecule, an inducer, binds to

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the repressor. Regulatory protein being an allosteric protein has two binding sites one to bind
DNA and another to bind small molecule (inducer). In general regulatory protein binds to
DNA and stop transcription, but when inducer is present it compete and get itself bind to
regulatory protein and thus altering the structure of regulatory protein so prevent its binding
to DNA.

Lac structural genes

Lactose is 𝛃𝛃-galactoside that use as a carbon source by E. coli.

lacZ: encodes for β-galactosidase : enzyme responsible for breakdown of lactose into glucose
and galactose. This enzyme also converts lactose to allolactose.

lacY: encodes for β-galactoside permease: responsible for facilitating the transport of lactose
across the bacterial cell wall.

lacA: encodes β-galactoside transacetylase: the actual function of this gene is not known, but
appears to have role in the detoxification of compounds by transferring acetyl-CoA to β-
galactosides

All three of these genes are transcribed as a single, polycistronic mRNA. Polycistronic RNA
contains multiple genetic messages each with its own translational initiation and termination
signals.

The lac Operon of E. coli

Lac Operon in E. coli : Negative Inducible Control :

E. coli can use lactose as its sole carbon source. Lac operon is an operon required for the
transport and metabolism of lactose in E. coli and some other enteric bacteria.

The lac operon includes the 3 structural genes –

• β galactosidase (lacZ gene)

• Galactoside permease (lacY gene)

• Thiogalactoside transacetylase (lacA gene)

β galactosidase cleaves lactose to galactose and glucose. Galactoside permease transports

lactose into the cell. Thiogalactoside transacetylase appears to modify toxic galactosides to
facilitate their removal from the cell. When E. coli is grown in medium where lactose is
added, β galactosidase, lac permease and transacetylase appear at the same time in cells.

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After lactose is transported into the cell, some of it is converted to allolactose by the few
molecules of β galactosidase. Allolactose is a rearranged lactose molecule and an inducer of
the lac operon. lacZ, lacY and lacA are coordinately expressed from one promoter called lacP
that directs expression of a polycistronic mRNA. lacP is located upstream of lacZ. lacI
encodes the lactose repressor protein and I located upstream of lacP. lacI has its own
promoter and is constitutively expressed. Lac repressor or lacI binds to the DNA at the lac
operator site called lacO. lacO is located between lacI and lacZ. In the absence of inducer, lac
repressor is bound to lacO, preventing expression of the operon. In the presence of inducer,
inducer binds to lac repressor and prevents repressor from binding to lacO, leading to
transcription of the operon.

Lac Operon in E. coli: Catabolite Repression:

E. coli and many other bacteria metabolize glucose in preference to lactose and other sugar as
a carbon source when they are offered a choice. They generally do this because directely
enters in the Glycolysis and requires less energy to metabolize than the other sugars. So, in
culture medium when E. coli find both sugars, it metabolize glucose first and genes that
participate in the metabolism of other sugars are repressed, this phenomenon is called as
glucose effect or Catabolite repression. Monod further suggested the diauxic growth of E coli
to this glucose effect and this catabolite repression results in positive control in response to
glucose.

This positive control is results due to the binding of a dimeric protein called the catabolite
activator protein (CAP) to a site that located 22 nucleotides upstream to the promoter of lac
genes (figure 3.2). But, RNA polymerase does not bind efficiently to many promoters unless
CAP is first bound to the DNA. Before CAP can bind to DNA, it must form a complex with a
modified nucleotide CAMP. Since, high level of glucose in the medium results in low level
of cAMP, low levels of cAMP mean very few cAMP-CAP complexes. Subsequently, RNA
polymerase has poor affinity for the lac promoter, and little transcription of the lac operon
takes place. When intracellular levels of glucose drop and other sugars must be metabolized,
levels of cAMP increase. Increased cAMP levels means there are more cAMP-CAP
complexes. cAMP-CAP binds to a specific site on the DNA that is located adjacent to the
promoter for the lac genes and adjacent to promoters controlling the expression of other sugar
metabolizing operons. This increase enhances the binding of RNA polymerase to the
promoter and increases transcription of the lac genes.

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Figure 3.2: Catabolic repression: (a) Absence of glucose, in the absence of glucose cAMP
levels increase, resulting in the formation of a cAMP–CAP complex, which facilitates
transcription. (b) Presence of glucose, in the presence of glucose cAMP levels decrease,
cAMP–CAP complexes are not formed and thus there is no transcription.

Tryptophan Operon – Negative Repressible Control:

Tryptophan is an important amino acid in living system and in bacteria a complex mechanism
is needed in its regulation. E. coli are capable for producing enzymes necessary for the
biosynthesis of amino acids. Monod worked on an amino acid tryptophan and enzyme
tryptophan synthetase and discovered that if tryptophan is present in sufficient quantity in the
culture medium for E. coli, the enzymes necessary for its synthesis are not produced. It is
energetically advantageous for bacteria to repress expression of genes involved in tryptophan
synthesis when tryptophan is present in the culture medium (figure 3.3 a &b). Thus, Trp
operon is an operon in bacteria which promotes the production of tryptophan when
tryptophan is not present in the growth medium. If the amino acid is present, then the operon
is repressed and biosynthetic enzymes are not produced.

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Figure 3.3 a: Tryptophan operon (when tryptophan present in the medium, it binds with the
repressor causing allosteric reaction and formed product binds to operator and thus
repressing transcription)

Further, investigation showed that a series of enzymes encoded by five contiguous genes
(trpE, trpD, trpC, trpB, and trpA, and encode tryptophan synthetase) on the E. coli
chromosome are involved in the conversion of chorismic acid to tryptophan. It also contains a
promoter where RNA polymerase binds and a repressor gene which synthesizes a specific
protein. This protein binds to the operator which then causes the transcription to be blocked.

Figure 3.3 b: Tryptophan operon (in the absence of Tryptophan, inactive regulator protein
cannot binds to operator, thus allowing transcription)

When tryptophan is abundant it binds to the trp repressor, causing a conformation change that
permits the repressor to bind to the trp operator and inhibit expression of the trp operon. The

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trp operator site overlaps the promoter, so binding of the repressor blocks binding of RNA
polymerase.

Riboswitches:

Till know we have understands that operons of bacteria contain DNA sequences (promoters
and operator sites) where the binding of small molecules induces or represses transcription.
Besides these, some mRNA molecules may contain regulatory sequences and the sequences
are called as riboswitches. They were first discovered in 2002 and now appear common in
bacterium, In Bacillus subtilis, for example, approximately 5 percent of this bacterium’s
genes are regulated by riboswitches. They are also found in archaea, fungi, and plants, and
may prove to be present in animals as well. These molecules may bind and affect gene
expression by the formation of secondary structures in the mRNA. The two important
domains within a riboswitch are the ligand-binding site, called the aptamer, and the
expression platform, which is capable of forming the terminator structure.

3.3.2 Control of gene expression in Eukaryotes

In eukaryotic organism, gene expression is under tight control of series of reaction and
checks in order to avoid any malfunction or over-function. So the genes are under very tight
control to express the required level of proteins, because minor changes in the expression
leads to lethal effects to the organism. To achieve the fine degree of control, eukaryotes
employed wide range of mechanism for altering the expression of genes. Thus,control of
gene expression in eukaryotes is controlled by a variety of mechanisms as followed –

1. Control at DNA level by Histone modifications and Chromatin Remodeling:

DNA is compacted into chromatin as part of its packaging strategy, this compacted form can
affect the ability of transcriptional regulatory proteins and RNA polymerase to find access to
specific genes for activation of transcription. Modification of the histones and of CpG
methylation most affect accessibility of the chromatin to RNA polymerases and transcription
factors.

a. Acetylation of Histones: One type of histone modification that affect chromatin

structure. These modifications can bring about either the activation or the repression
of transcription. Acetylation is catalyzed by histone acetyltransferase enzymes
(HATs). A histone is wrapped twice around the nucleosome. They have positive
lysine residues on their N-termini and are strongly attracted to DNA because of its
highly negative phosphate backbone. If acetyl groups are added to the lysine 16 in the
tail of the H4 histone prevents the formation of the 30-nm chromatin fiber causing the
chromatin to be in an open configuration and available for transcription. The charge is
neutralized which makes it possible for chromatin remodeling protein to nudge the

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nucleosomes so that the TATA box is exposed. With TATA box exposed, TATA
binding factor and transcription factor can bind to it, allowing RNA polymerase to
bind to the pre-initiation complex and start transcription; other enzymes called
deacetylases (histone deacetylases or HDACs) strip acetyl groups from histones and
restore chromatin structure, which represses transcription because the deacetylase will
remove acetyl groups from lysine on the N-termini of histones. This would cause the
positive charge of the lysine and the negative charge on the DNA to become strongly
attracted to each other and the histones would bind tightly.

b. Methylation of Histones: A common modification is the addition of three methyl

groups to lysine 4 in the tail of the H3 histone protein, abbreviated H3K4me3. This
modification is frequently found in the transcriptionally active genes in eukaryotes.
Recent studies suggested that NUFR and certain proteins binds to the H3 histone tail
and allowing the transcription to take place.
c. Methylation of DNA: Another type of change in chromatin that plays a role in gene
regulation is the methylation of cytosine bases, which yields 5-methylcytosine.
Methylation occurs most often in symmetrical CG sequences (CpG, where p
represents the phosphate group in the DNA backbone, called CpG island) in small
percentage of newly synthesized DNA. DNA methylation prevents the binding of the
transcriptional machinery and it’s associated with transcriptional silencing. This
methylation have inverse relationship with the gene expression, therefore
transcriptionally inactive genes possess higher levels of methylated DNA than
transcriptionally active genes. There are number of evidence to support the
observation i.e., inactivated X chromosome in mammalian female cells are often
heavily methylated and another example is that the methylation patterns are tissue
specific and once established, are heritable for all cells of that tissue.

2. Control at Transcription Initiation:

The regulation of gene expression at the level of transcription is most important and primary
mode of control of eukaryotic gene expression. Transcription of the different classes of RNAs
in eukaryotes is carried out by three different polymerase. Such as RNA pol I synthesizes the
rRNA. RNA pol II synthesizes the mRNA and some snRNA. RNA pol III synthesizes the 5S
rRNA and the tRNA. Transcription regulation in a eukaryotic cell is mediated by
transcription factor, other than the general transcription factors, which recognized and bind to
short regulatory DNA sequences associated with the gene. These sequences are also called
cis-acting elements and the protein transcription factors that bind to these elements are also
known as transacting factors. These factors which regulate specific gene transcription do so
by interacting with the proteins of the transcription initiation complex and may either
increase or decrease the rate of transcription of the target gene. Many transcription factors
bind to control elements located upstream within a few hundred base parirs of the protein

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coding gene. The SPI box (the SPI box has the core sequence GGGCGG, and binds to
transcription factor SP1 which then interacts with one of the TAFII proteins that bind to TBP
to form TFIID) and CAAT box are examples of such regulatory elements found upstream of
most protein coding genes, but some upstream regulatory elements are associated with only a
few genes and are responsible for gene specific transcriptional regulation. Besides this, many
repressor protein that inhibits the transcription of specific gene in eukaryotes may bind either
to control elements near to the target gene or to silencers that may be located a long distance
away. The repressor protein may inhibit transcription of the target genes directly (example in
the mammalian thyroid hormone receptor which, in the absence of thyroid hormone,
represses transcription by blocking activation). However, other repressors inhibit
transcription by blocking activation or by interfering with the function of an activator protein
required for efficient gene transcription.

3. Control at mRNA Splicing – Alternate Splicing:

Most eukaryotic pre – mRNA contain multiple introns (non coding DNA). The process of
excising the sequences in RNA that corresponds to introns and joining of sequences
corresponding to exon is called RNA. Splicing is done by spliceosomes, ribonucleoprotein
complexes that can recognize the two ends of the intron, cut the transcript at those two points,
and bring the exons together for ligation. Cajal bodies within the nucleus are centers of post-
transcriptional modification of snRNAs and snRNPs assembly. Alternative RNA splicing is a
mechanism that allows different protein products to be produced from one gene when
different combinations of exons are combined to form the mRNA. Use of alternative splice
sites in the same pre – mRNA allows the synthesis of different proteins from the same gene
or can function as an on – off switch to make or not make a functional protein. Drosophila
sex determination, T-antigen gene of the mammalian virus SV40 and human genome
provides the best example of a regulated alternative splicing. Further, in trans-splicing (found
in few eukaryotic organisms), exons from two separate RNA transcripts are spliced together
to form a mature mRNA molecule.

4. Control at mRNA Stability:

Once mRNAs are produced, they get exported from the nucleus to the cytoplasm. They are
given two protective "caps" that prevent the ends of the strand from degrading during its
journey. The 5' cap, which is placed on the 5' end of the mRNA, is usually composed of a
methylated guanosine triphosphate molecule (GTP). The poly-A tail, which is attached to the
3' end, is usually composed of a long chain of adenine nucleotides. These changes protect the
two ends of the RNA from exonuclease attack. Within the cytoplasm they are translated until
degradation. Long – lived mRNA give rise to more polypeptides than short – lived mRNAs.
Therefore mRNA life time also can be targeted as an important point for regulation of gene
expression. The longevity of mRNA is influenced by some factors such as: The sequence of
the 3’ UTR preceding the poly – A tail also affects mRNA stability. Some short – lived

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mRNAs have many repeated sequences AUUUA in this region, referred to as an AU rich
element. Further, the concentration of some metabolites, such as hormones, can also
influence mRNAs longevity.

5. Control at Initiation of Translation:

Regulation of gene expression may also occur at the level of translation, but the regulation is
less efficient in comparison to the transcriptional regulations. Control mechanism at initiation
of translation either prevent the synthesis of protein or controls the amount of protein
produced from a given mRNA. An example of translational control is proteins involved in
iron metabolism. The level of ferritin is regulated in response to the iron supply. When iron
concentration is low, translation of ferritin mRNA is reduced, while when iron concentration
is high, ferritin synthesis increases.

6. Post – Translational Control:

Post translational control of gene expression can be defined as a regulation process in which
protein structure is modified and as a result protein function is modified. These modifications
involves chemical modification of amino acids or alteration of the order of amino acids in the
polypeptide backbone. The most common chemical modification of amino acids include
phosphorylation, glycosylation and ubiquitiation.

7. RNA Editing:

RNA editing can be best define as a process in which the RNA sequence is modified, so the
mature RAN differ from that encoded by the genomic sequence or in other words RNA
editing is the name given to several reactions whereby the nucleotide sequence on an mRNA
molecule may be changed by mechanism other than the RNA splicing. An example of RNA
editing in human is apolipoprotein B mRNA. Liver cells produce apolipoprotein B100
without any RNA editing, but in the cells of intestine, RNA editing causes the conversion of
single C residue to U and this changes the codon for glutamine (CAA) to a termination codon
(UAA), which further results in shorter apolipoprotein B48(48% of the size of apolipoprotein
B100). There are two RNA editing mechanisms which include: a site specific base
modification and insertion-deletion editing.

a. Site specific editing (substitution editing): In site specific editing (include transversion and
transition in all classes of RNA), a cytosine residue within the RNA is changed into uridine
by deamination. For example, deamination of adenosine to produce inosine. Since inosine
(inosine behave like guanosine) can base pair with cytosine, this induces point mutation
which alter the protein sequence.

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b. Insertion-Deletion editing: This type of editing reported in the mitochondrial RNA of some
Protozoans. In this mechanism uridines are inserted into the transcripts as found in the
mitochondrial mRNA of trypanosomes. These insertions completely shift the reading frame
and give rise to totally different proteins.

8. Regulation by miRNA:

MicroRNAs (miRNAs) or short RNA molecule that are about 20-22 nucleotide long. The
miRNAs are made in the nucleus as longer pre-miRNAs. These pre-miRNAs are chopped
into mature miRNAs by a protein called Dicer. There are hundreds of genes encoding
miRNA in eukaryotes and most of them are involved in developmental regulation. These
microRNAs regulate gene expression through at least four distinct mechanisms: (1) cleavage
of mRNA, (2) inhibition of translation, (3) transcriptional silencing, or (4) degradation of
mRNA.

3.4 SUMMARY

In this unit, we learnt various mechanisms for control of gene expression in prokaryotes as
well eukaryotes. Jacob and Monod 1961, proposed a scheme for induction and repression for
which they awarded nobel prize in 1965. Genes in bacterial cells are typically clustered into
an operons group of functionally related structural genes and the sequences that control their
transcription. Structural genes in an operon are transcribed together as a single mRNA
molecule. In negative control, a repressor protein binds to DNA and inhibits transcription and
in positive control, an activator protein binds to DNA and stimulates transcription. Further, In
inducible operons, transcription is normally off and must be turned on; in repressible operons,
transcription is normally on and must be turned off. The lac operon of E. coli is a negative
inducible operon. While the, trp operon of E. coli is a negative repressible operon that
controls the biosynthesis of tryptophan. Attenuation causes premature termination of
transcription. riboswitches in mRNA molecules induce changes in the secondary structure of
the mRNA, which affects gene expression.

But this process is somewhat different in eukaryotic. Eukaryotic cells differ from bacteria in
several ways that affect gene regulation, including, in eukaryotes, the absence of operons and
the presence of chromatin & nuclear membrane. Chromatin play an important role in the gene
expression and during transcription, chromatin structure may be altered by the modification
of histone proteins, including acetylation, phosphorylation, and methylation. Besides these,
the repositioning of nucleosomes and the methylation of DNA also affect transcription or
may contribute in gene regulation. The Epigenetic effects are resulted due to the DNA
methylation and alterations to chromatin structure. Further, the initiation of eukaryotic
transcription is controlled by general transcription factors that assemble into the basal
transcription apparatus and by transcriptional activator proteins that stimulate normal levels

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of transcription by binding to regulatory promoters and enhancers. The expression of gene in

eukaryotic cells may also influenced by RNA processing. The 5′ cap, the coding sequence,
the 3′ UTR, and the poly(A) tail are important in controlling the stability of eukaryotic
mRNAs. Proteins binding to the 5′ and 3′ ends of eukaryotic mRNA can affect its translation.
. Posttranscriptional gene regulation includes alternative splicing of nascent RNA, RNA
transport, or changes in mRNA stability. Alternative splicing increases the number of gene
products encoded by a single gene. Posttranscriptional gene regulation at the levels of
translation and protein stability also affect the levels of active gene product. RNA-induced
gene silencing is a posttranscriptional mechanism of gene regulation that affects the
translatability or stability of mRNA as well as transcription.

Thus from this chapter we come to understand that, the aim of regulation is to control the
gene expression to only when they are needed. For example, in prokaryotes the catabolic
machinery needed to metabolize lactose only needs to be available when lactose is present.
We also saw that regulation of gene expression in eukaryotes is crucial for an essential
multicellular organism to develop harmoniously according to a pre – determined genetic
program. The eukaryotic cell structure provides possible control for gene expression at many
levels such as chromatin structure, transcription initiation, at RNA level and post – translation
level.

3.5 GLOSSARY

3' UTR: 3' untranslated region; region just downstream of the protein-coding region in an
RNA molecule that is not translated.

5' cap: a methylated guanosine triphosphate (GTP) molecule that is attached to the 5' end of a
messenger RNA to protect the end from degradation.

5' UTR: 5' untranslated region; region just upstream of the protein-coding region in an RNA
molecule that is not translated.

Activator: protein that binds to prokaryotic operators to increase transcription.

Catabolite activator protein (CAP): protein that complexes with cAMP to bind to the
promoter sequences of operons which control sugar processing when glucose is not available.

Cis-acting element: transcription factor binding sites within the promoter that regulate the
transcription of a gene adjacent to it.

Cistron: a section of a DNA or RNA molecule that codes for a specific polypeptide in
protein Synthesis.

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Codon: Sequence of three nucleotides in DNA or mRNA that specifies a particular amino
acid during protein synthesis; also called triplet.

Consensus sequence: The nucleotides or amino acids most commonly found at each position
in the sequences of related DNAs, RNAs or proteins.

Dicer: enzyme that chops the pre-miRNA into the mature form of the miRNA.

DNA methylation: epigenetic modification that leads to gene silencing; a process involving
adding a methyl group to the DNA molecule.

Enhancer: segment of DNA that is upstream, downstream, perhaps thousands of nucleotides

away, or on another chromosome that influence the transcription of a specific gene.

Epigenetic: heritable changes that do not involve changes in the DNA sequence.

Eukaryotic initiation factor-2 (eIF-2): protein that binds first to an mRNA to initiate
translation.

Exon: Segment of a eukaryotic gene that reaches the cytoplasm as part of a mature mRNA,
rRNA or tRNA.

Gene expression: Overall process by which the information encoded in a gene is converted
into an observable phenotype.

Gene: Physical or functional unit of heredity, which carries information from one generation
to the next.

Genetic code: The set of rules whereby nucleotide triplets or codon in DNA or RNA specify
amino acid in proteins.

Genome: Total genetic information carried by a cell or organism.

Glycosyl transferase: An enzyme that forms a glycosidic bond between a sugar residue and
an amino acid side chain of a protein or a residue in an existing carbohydrate chian.

Histone acetylation: epigenetic modification that leads to gene silencing; a process involving
adding or removing an acetyl functional group.

Inducible operon: operon that can be activated or repressed depending on cellular needs and
the surrounding environment.

Initiation complex: protein complex containing eIF-2 that starts translation.

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Intron: Part of a primary transcript that is removed by splicing during RNA processing and is
not included in the mature, functional mRNA, rRNA or tRNA.

microRNA (miRNA): small RNA molecules (approximately 21 nucleotides in length) that

bind to RNA molecules to degrade them.

Monocistronic: Describing a type of messenger RNA that can encode only one polypeptide
per RNA molecule. In eukaryotic cells virtually all messenger RNAs are monocistronic.

Negative regulator: protein that prevents transcription.

Operator: region of DNA outside of the promoter region that binds activators or repressors
that control gene expression in prokaryotic cells.

Operon: In bacterial DNA, a cluster of contiguous genes transcribed from one promoter that
gives rise to a polycistronic mRNA.

Poly-A tail: a series of adenine nucleotides that are attached to the 3' end of an mRNA to
protect the end from.

3.6 SELF ASSESSMENT QUESTION

3.6.1 Objective type questions

Q1. To which region of a gene does an RNA polymerase bind to initiate transcription?

(i) 5’ UTR (ii) 3’ UTR

(iii) CDS (iv) Promoter

Q2. Which of the following processes generates multiple transcripts from the same gene?

(i) Riboswitching (ii) Alternative splicing

(iii) Short-peptide translation (iv) All of these

Q 3. Which of the following processes involves metabolite-sensing in non-coding portions of

mRNAs to control gene expression?

(i) Alternative polyadenylation (ii) Riboswitching

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(iii) Adenosine methylation (iv) Short-peptide

translation

Q4. A genomic DNA possesses functioning units, a group of genes under the influence of
promoters known as

(i) genes (ii) operons

(iii) anticodon (iv) codon

Q5. There are these many histones in the core of a nucleosome

(i) 8 (ii) 6

(iii) 4 (iv) 2

Q6. In eukaryotes and bacteria, the most common form of regulation is

(i) promoter control (ii) translation control

(iii) repressor control (iv) transcriptional control

Q7. In a bacterial operon, which is located downstream of the structural genes?

(i) operator (ii) inducer

(iii) promoter (iv) regulatory gene

Q8. The lac operon consists of ____ structural genes.

(i) 1 (ii) 2

(iii) 3 (iv) 4

Q9. Which of the following acts as an inducer in the lac operon?

(i) glucose (ii) tryptophan

(iii) lactose (iv) galactose

In bacteria, mRNAs bound to small metabolites are called

(i) euchromatin (ii) riboswitches

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(iii) heterochromatin (iv) nucleosome

3.6.1 Answers: 1-(iv), 2- (ii), 3- (ii), 4- (ii), 5-(i), 6- (iv), 7-(iv), 8- (ii), 9-(iii), 10- (ii).

3.6.2 Short notes

Q1. What are promoters and operators?

Q2. Mention the functions of lac operon genes.

Q3. What is RNA interference?

Q4. What are inducible genes and repressible genes?

3.7 REFERENCES
1. Babitske, P., Baker, C.S., & Romeo, T. (2009) Regulation of translation initiation by
RNA binding proteins. Annu. Rev. Microbiol. 63, 27–44.
2. Berezikov, E. (2011) Evolution of microRNA diversity and regulation in animals.
Nat. Rev. Genet. 12, 846–860.
3. Conaway, R.C. & Conaway, J.W. (2011) Function and regulation of the Mediator
complex. Curr. Opin. Genet. Dev. 21, 225–230.
4. Fabian, M.R., Sonenberg, N., & Filipowicz, W. (2010) Regulation of mRNA
translation and stability by microRNAs. Annu. Rev. Biochem. 79, 351–379.
5. Groppo, R. & Richter, J.D. (2009) Translational control from head to tail. Curr. Opin.
Cell Biol. 21, 444–451.
6. Jacob, F. & Monod, J. (1961) Genetic regulatory mechanisms in the synthesis of
proteins. J. Mol. Biol. 3, 318–356.
7. Keene, J.D. (2007) RNA regulons: coordination of posttranscriptional events. Nat.
Rev. Genet. 8, 533–543.
8. Krol, J., Loedige, I., & Filipowicz, W. (2010) The widespread regulation of
microRNA biogenesis, function and decay. Nat. Rev. Genet. 11, 597–610.
9. Osterberg, S., del Peso-Santos, T., & Shingler, V. (2011) Regulation of alternative
sigma factor use. Annu. Rev. Microbiol. 65, 37–55.
10. Pasquinelli, A.E. (2012) MicroRNAs and their targets: recognition, regulation and an
emerging reciprocal relationship. Nat. Rev. Genet. 13, 271–282.
11. Rinn, J.L. & Chang, H.Y. (2012) Genome regulation by long noncoding RNAs. Annu.
Rev. Biochem. 81, 145–166.
12. Roth, A. & Breaker, R.R. (2009) The structural and functional diversity of metabolite-
binding riboswitches. Annu. Rev. Biochem. 78, 305–334.

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13. Shahbazian, M.D. & Grunstein, M. (2007) Functions of sitespecific histone

acetylation and deacetylation. Annu. Rev. Biochem. 76, 75–100.
14. Struhl, K. (1999) Fundamentally different logic of gene regulation in eukaryotes and
prokaryotes. Cell 98, 1–4.
15. Talbert, P.B. & Henikoff, S. (2010) Histone variants—ancient wrap artists of the
epigenome. Nat. Rev. Mol. Cell Biol. 11, 264–275.
16. Werner, F. & Grohmann, D. (2011) Evolution of multisubunit RNA polymerases in
the three domains of life. Nat. Rev. Microbiol. 9, 85–98.
17. Zhou, Q., Li, T., & Price, D.H. (2012) RNA polymerase II elongation control. Annu.
Rev. Biochem. 81, 119–143.
18. Struhl, K. (1999). Fundamentally different logic of gene regulation in eukaryotes and
prokaryotes. Cell 98, 1–4

3.8 SUGGESTED READINGS

Hartl, D.L and Jones, E.W (1998) Genetics:Principles and Analysis, Carnegie Mellon
University Jones and Bartlett Publishers.

Gardner, T.S., Cantor, C.R., & Collins, J.J. (2000) Construction of a genetic toggle switch in
Escherichia coli. Nature 403, 339–342.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2009) Molecular
Biology of The Cell, Fifth Edition. Published by Garland Science, Taylor & Francis Group.

Nelson, D.L and Cox, M.M (2013) Principle of biochemistry, sixth edition, publisher W. H.
Freeman and Company.

Pierce, B.A. (2012). Genetics A Conceptual Approach, Fourth Edition Southwestern

University W. H. Freeman and Company.

Molecular Biology of the Gene, Fifth Edition. Watson, Baker, Bell, Gann, Levine, Losick.
Principles of Biochemistry, Fourth Edition. Lehninger.

3.9 TERMINAL QUESTIONS

Q1. What is the difference between positive and negative control? What is the difference
between inducible and repressible operons?

Q2. Briefly describe the lac operon and how it controls the metabolism of lactose.

Q3. How does bacterial gene regulation differ from eukaryotic gene regulation? How are they
similar?

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Q4. Outline the role of alternative splicing in the control of sex differentiation in Drosophila.

Q5. What role does RNA stability play in gene regulation?

Q6. What is catabolite repression? How does it allow a bacterial cell to use glucose in
preference to other sugars?

Q7. Write an essay on regulation of gene activity in eukaryotes.

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BLOCK-2
BIOTECHNOLOGY
MOLECULAR BIOLOGY AND BIOTECHNOLOGY (MSCBOT-604)

UNIT-4: BASIC CONCEPTS, PRINCIPLES AND SCOPE OF

BIOTECHNOLOGY

Contents:
4.1 Introduction
4.2 Objectives
4.3 Basic concept of biotechnology
4.3.1 Various stages of biotechnology development
4.4 Definitions
4.5 Types of biotechnology
4.6 Applications of biotechnology
4.7 Various techniques in biotechnology
4.8 Plant biotechnology
4.9 Methods in plant biotechnology
4.9.1 Plant Tissue Culture
4.9.2 Genetic engineering
4.9.3 Molecular Assisted Breeding (MAB)/Marker Assisted Selection (MAS)
4.10 Applications of plant biotechnology
4.10.1 Crop improvement
4.10.2 Genetic Transformation
4.10.3 Pharmaceuticals
4.10.4 Industrial
4.11 Scope of biotechnology
4.12 Summary
4.13 Self-assessment questions
4.13.1 Multiple choice questions
4.13.2 Fill in the blanks
4.14 References
4.15 Suggested readings
4.16 Terminal questions
4.16.1 Short answer type questions
4.16.2 Long answer type questions

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4.1. OBJECTIVES
• After reading this unit you would be able to:
 Describe the basic concepts of biotechnology;
 Define and enumerate the principles of plant biotechnology;
 Discuss the scope of Biotechnology

4.2. INTRODUCTION
The use of living organisms to create technologies and goods for human improvement and
sustainable development is known as biotechnology. It is a branch of applied biology and a
synthesis of biology and technology. The idea of biotechnology first emerged in the 19th century
when plant and animal cells, along with their component parts, were cultivated in vitro to
produce desired goods. Since then, microbes have been employed in the mass manufacture of
biochemicals, processing of food, and environmental cleanup.
The main applications of biotechnology are in the areas of therapeutics, diagnostics, food
processing, bioremediation, waste treatment, energy production, technology, medicine and other
fields that require bio-products. It is also used to improve agriculture through the use of
genetically modified crops. Recombinant DNA technology has made it feasible to produce
microbes, plants, and animals that possess superior and novel capabilities. Genes from one
organism have been added to another to generate genetically modified organisms, or GMOs. For
tolerance to high temperatures, salt stress, and resistance to disease and insect attack, plants have
been genetically engineered. These approaches have shown to be very effective in raising crop
plant production and lowering post-harvest losses. The use of biotechnology has enabled the
development of food plants with increased nutritional value and a decreased dependency on
chemical pesticides (pest-resistant crops). The current Unit introduces you to the idea of
biotechnology and informs you about its scope, applications and several plant biotechnology
techniques and their uses.

4.3. BASIC CONCEPT OF BIOTECHNOLOGY

Biotechnology is NOT brand-new. Man has been utilizing living things to better his life ever
since the beginning of time. Man has used plants and animals for various purposes since the
beginning of time (about 8000–4000 B.C.). It is important to understand the basic concept of
biotechnology in order to understand its principles and processes.
Biotechnology is the use of living systems and organisms to produce or develop useful products.
It can also refer to "any technological" application that uses biological systems, live organisms,
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or their derivatives to produce, alter, or produce goods for a specific purpose. The application of
biotechnology in agriculture, food production, and medicine has been practiced by humans for
thousands of years.
The development of pharmacological treatments and diagnostic procedures, as well as new and
diversified sciences like genomics, recombinant gene technologies, applied immunology, and
others, have all been included into the field of biotechnology in the late 20th and early 21st
centuries. Over time, there were several developments made in the field of biotechnology.
4.3.1. Various stages of biotechnology development
The advancement of biotechnology falls under the following categories:
• Ancient biotechnology (8000–4000 BC): Early biotechnology history is associated with
the domestication of animals and the cultivation of plants.
• Classical biotechnology (2000 BC; 1800–1900 AD): This period of biotechnology was
founded on the use of microbes in fermentation, food production, and medicine. Genetics
developed during this time (1900–1953), and DNA study started (1953–1976).
• Modern biotechnology (1977): During this time, genetic engineering was used to alter
the genetic material of living things.
Ancient biotechnology
Before the year 1800, activities like the domestication of animals for milk or meat and the
cultivation of plants like rice, barley, and wheat as the primary source of sustenance were
considered as biotechnological advances. Animals and plants have been bred in an effort to
better them by introducing desirable traits.
Classical biotechnology
Microorganisms were employed to produce foods like bread, cheese, and beverages after
carefully breeding plants and animals. Leguminous crop rotation, vaccines, and animal-drawn
machinery all became common in the late eighteenth and early nineteenth centuries. A turning
point in biology occurred around the end of the nineteenth century. Mendel's genetic research
was completed, microorganisms were identified, and Koch, Pasteur,
and Lister founded research centers to study fermentation and other
microbial activities. Edward Jenner, who is regarded as the father of
vaccination science, established the effectiveness of vaccination in
preventing smallpox in 1796. Louis Pasteur gave the first description
of the scientific evidence for fermentation in the late 1800s. He put out
the germ theory, which holds that microbes are crucial to the
fermentation process. Since ancient times, honey and soybean curds
have been utilized as natural antibiotics and have been proven to be
successful in treating wounds. Proteins and enzymes were first
identified in 1830.

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The term "Biotechnology" was first used by Hungarian engineer Karl Ereky in 1919. He defines
biotechnology as the use of living organisms to the production of valuable resources or products.
In other words, it is the technology that uses biological mechanisms, systems, or processes to
produce products that enhance human life.
Karl Ereky
Beginning in the early 20th century, biotechnology started to connect business and agriculture.
For the rapidly expanding vehicle industry, fermentation techniques were created during World
War-I, in 1920, Chaim Weizman produce valuable chemical compounds using biological
methods. Utilizing a bacterium called Clostridium acetobutylicum, he produced butanol from
starch. Later, acetone from starch and biotechnology techniques like fermentation was developed
for use in the automotive paint industry. The production of penicillin began with the outbreak of
World War-II. In 1929, Alexander Fleming produced antibiotic penicillin from cultures of
Penicillium notatum. The penicillin was helpful in treating soldiers who were injured World
War-II. After that the focus of biotechnology shifted to drugs such as development of antibiotics,
fermentation techniques, and work with microorganisms etc.
Modern biotechnology
Currently, modern biotechnology is applied in numerous industries, such as agriculture,
bioremediation, food processing, and energy production. In forensics, the use of DNA
fingerprinting is increasingly widespread. Cloning vectors with the chosen gene already present
allows for the production of insulin and other medications. Cellular and molecular technologies
started to develop later in the 1960s and 1970s. Research on DNA was undertaken between 1953
and 1976.To produce valuable products, cells and biological substances like DNA, RNA, and
proteins were used. The real biotechnology was initiated with the production of Insulin in 1970.
A similar discovery was made in 1970 regarding the first restriction enzyme. By making this
finding, the researchers were able to introduce foreign genes into bacteria. The development of
recombinant DNA technology was based on this, and it is regarded as the beginning of modern
biotechnology. Living things' DNA was manipulated, and substances produced from GMOs were
applied to the alteration of molecules. Dolly, the sheep was cloned in 1977 by scientists in
Scotland; later, Polly the sheep was cloned using nuclear transfer technique since it possessed a
human gene expressing Factor IX, a protein important in preventing hemophilia. In the middle to
late 1970s, saw the founding of businesses like Genentech, Amgen, Biogen, Cetus, and Genex.
They produced genetically modified materials mainly for environmental and medical purposes.
Stem cell research has attracted the attention of molecular biologists. Antibody analysis was
discovered to be another useful tool in 1999. Modern biotechnology is used in therapeutics,
primarily for the discovery, development, and production of novel medications, and in
diagnostics, for protein- and nucleic acid-based tests. It has mostly been employed in the
environmental field to treat waste and prevent pollution. To produce plants that are resistant to
pests, weeds, and plant diseases, genetic engineering is being applied in agriculture. Thus
modern biotechnology makes significant contribution in improving the lives of humans. A brief
overview of the biotechnology's past occurrences has been provided in Table 4.1.

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4.4. DEFINITIONS
Biotechnology is the combination of the two words "bio" for life and "technology," which refers
to the application of knowledge for practical use, i.e., the use of living things to make or improve
products.

A "Janus-faced" description of biotechnology has been used. By implication, two sides are
present. Techniques enable the transfer of genes from one organism to another by manipulating
DNA on the one hand. On the other hand, it makes use of recent technologies whose effects have
not yet been thoroughly investigated and should be approached with caution.

• “All area of work by which product are produced from raw materials with the aid of
living things”. -Karl Ereky
• Biotechnology is defined as the “integrated use of biochemistry microbiology and
engineering science in order to achieve technological application of the capabilities of
microorganism. Cultured tissue cell and part’s thereof”.
-European Federation of
Biotechnology
• “A technology using biological phenomenon for copying and manufacturing various kind
of useful substances”. -Japanese
biotechnology
• “The controlled used of biological agent such as micro-organism or cellular components
for beneficial use.” -U.S. National science foundation

Table 4.1 An overview of biotechnology's past occurrences.

Period Time/ Year Historical Events
Pre-1800 6000 BC Yeast was used to prepare beer
4000 BC In Egypt, yeast was used to prepare bread.
420 BC Greek philosopher Socrates found that similar characteristics are
found between parents and their offspring.
320 BC Greek philosopher Aristotle gave the concept that characters get
inherited from father.
1000 AD Theory of abiogenesis, or spontaneous generation was proposed.
1630 William Harvey discovered the circulation of blood in the body.
1660–1675 Marcello Malpighi found blood circulation in capillaries using a
microscope and found that the brain is connected to the spinal
cord by bundles of fibers which form the nervous system.

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1673 Antonie van Leeuwenhoek identified microorganisms such as

protozoa and bacteria, and suggested that micro-organisms play
an active role in fermentation.
1701 Giacomo Pylarini found that the administration of smallpox
prevents its occurrence later in life. The procedure was termed
'vaccination'.
1800–1900 1809 Nicolas Appert invents a technique using heat to sterilize food.
1856 Justus von Liebig, Pasteur (1822–1895) suggested that microbes
are responsible for fermentation.
1859 Charles Darwin (1809–1882) speculated 'Natural Selection'.
1863 The method of pasteurization was discovered by Pasteur.
Heinrich Anton de Bary found that fungus was responsible for
potato blight.
1865 Mendel suggested ‘the Laws of Heredity’.
1868 Johannes Friedrich Miescher separated Nuclein (a compound
made of nucleic acid) from pus cells.
1870 Walther Flemming discovered Mitosis.
1871 Koch investigated anthrax and explored techniques to identify
culture and stain micro-organisms.
1880 Louis Pasteur explored weakened (attenuated) strains of micro-
organisms that might not be virulent.
1881 Koch explained techniques for harvesting bacterial colonies on
potato slices, gelatin and agar medium.
1884 Gram described the differential staining technique for cellular
peptidoglycan-containing bacteria now known as Gram staining.
1900-1953 1900 Mendel's work was revived by de Vries, von Tschermak and
Correns.
1902 Sutton found that chromosomes (paired) contain certain elements
which are transferred from one generation to another.
1905 Edmund Beecher Wilson and Nettie Stevens demonstrated that a
single Y chromosome determines maleness, while two copies of
the X chromosome decide femaleness.
1905-1908 William Bateson and R C Punnett found that several genes alter
or modify the action of other genes.
1906 Paul Erlich also investigated atoxyl compounds and discovered
the important features of Salvarsan (the first chemotherapeutic
agent).
1907 Thomas Hunt Morgan revealed that chromosomes have a defined
role in heredity. He discovered mutation theory into fruit flies.
1909 Wilhelm Johannsen used the word 'gene' for the
carrier/transporter of heredity. He also coined the terms
'genotype' and 'phenotype'.

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1910 Morgan demonstrated that genes are present on chromosomes.

1911 Morgan established the separation of certain inherited features
that are generally linked to the separation/breaking of
chromosomes during the process of cell division.
1912 William Lawrence Bragg discovered the application of X-rays in
the determination of the molecular structure of crystalline
substances (Crystallography).
1918 Herbert M Evans found that human genetic material is made up
of 48 chromosomes.
1926 Hermann Joseph Muller discovered that X-rays are responsible
for genetic mutations in fruit flies.
1928 Frederick Griffiths observed the 'transforming principle' by which
a rough type of bacterium is transformed to a smooth type.
1938 Proteins and DNA were studied by means of X-rays. The term
'molecular biology' was coined.
1941 George Wells Beadle and Edward L Tatum proposed 'one gene,
one enzyme' theory.
1943 Canadian scientist Oswald Theordore Avery discovered that
DNA is the carrier of genes.
1943-53 Cortisone, a steroid hormone was considered as the first biotech
product.
1944 Selman Abraham Waksman explored streptomycin, an antibiotic
active against TB.
1945-1950 Animal cell cultures were harvested for the first time in
laboratories, giving birth to the field of animal tissue culture.
1947 Barbara McClintock first demonstrated 'transposable elements'
known as 'jumping genes' with the capability to move (or jump)
from one site on the genome to another site.
1950 Erwin Chargaff discovered that the same levels of adenine and
thymine are present in DNA, as are the same levels of guanine
and cytosine. These associations were later named 'Chargaff's
rules'.
1952 George Otto Gey created a cell line known as HeLa from a
human cervical carcinoma.
1953-1976 1953 Watson and Crick's article based on unfolding the double-helix
structure of DNA was published in the journal, Nature.
1953–1976 The discovery of the structure of DNA revolutionized molecular
biology and genetics.
1957 Crick and Gamov studied 'central dogma’, demonstrating how
DNA functions to construct protein.

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1958 Arthur Kornberg created DNA in a test tube for the first time.
The mechanical protein sequencer, the Moore–Stein amino acid
analyzer, was developed.
1959 François Jacob and Jacques Lucien Monod documented concept
of genetic regulation. They explained the concept of 'repressor'
and 'operon'.
1960 A French researcher discovered messenger RNA (mRNA).
1961 Francois Jacob and Jacques Monad gave the concept of Operon.
1962 Watson and Crick were awarded the Nobel Prize in Physiology or
Medicine with Maurice Wilkins.
1963 Samuel Katz and John F Enders developed the first vaccine for
measles.
1964 Enzyme reverse transcriptase was discovered.
1966 Marshall Warren Nirenberg, J Heinrich Matthaei and S Ochoa
reported that a genetic sequence of three nucleotide bases (called
codons) decides each of 20 amino acids.
1967 Arthur Kornberg reported a study using single stranded natural
viral DNA.
1969 An enzyme is synthesized in vitro conditions.
1970 Virologists Peter H Duesberg and Peter K Vogt identified the
first oncogene in a virus.
1967-1971 Maurice Hilleman made the first American vaccine for mumps.
The first vaccine for rubella was developed.
1972 Paul Berg, a biochemist, utilized a restriction enzyme to cut DNA
into fragments. He employed a ligase enzyme to join two DNA
strands concurrently to form a hybrid circular molecule.
1973 Bruce Nathan Ames, a biochemist developed an investigation to
distinguish chemicals that damage DNA. Later, the Ames test
became extensively used to identify cancer-causing substances.
1974 The first vaccine for chicken pox was developed.
1975 Colony hybridization and Southern blotting were explored for
identifying specific DNA sequences. The first monoclonal
antibodies were prepared.
1976 J Michael Bishop and Harold Varmus established the presence of
cancer-causing genes called oncogenes on animal chromosomes.
The NIH published the first guidelines for rDNA research.
1977– 1977 With the advent of genetic engineering it was possible to produce
(Modern human protein in bacteria for the first time.
biotechnology) R Austrian and his coworkers developed the first vaccine for
pneumonia.

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1978 Herbert W Boyer synthesized synthetic human insulin by

introducing the insulin gene into the bacterium Escherichia coli.
Louise Brown, the first test-tube baby, was born in the UK.
The first vaccine for meningococcal meningitis was developed.
1980 Kary Mullis and coworkers established a tool for multiplying
DNA sequences in vitro using the polymerase chain reaction
(PCR).
1982 US Food and Drug Administration (FDA) allowed the first
genetically engineered drug in the form of human insulin
produced by bacteria.
Michael Smith at the University of British Columbia, Vancouver,
established a procedure for producing precise amino acid changes
anywhere in a protein.
1983 The first genetically modified (transgenic) plant is formed.
1984 The DNA fingerprinting technique was discovered.
The first genetically engineered vaccine was discovered for
hepatitis B.
1985 The NIH published guidelines for performing experiments in
gene therapy on humans.
Genetically engineered plants resistant to viruses, insects and
bacteria were field-tested for the first time.
1986 The NIH sanctioned guidelines for executing trials of gene
therapy on humans.
The automated DNA sequence was discovered in California.
1987 Calgene Inc. obtained a patent for the tomato polygalacturonase
DNA sequence, which was later used to synthesize an antisense
RNA sequence that can further extend the shelf life of fruit.
Maynard Olson and colleagues at Washington University
discovered yeast artificial chromosomes, which are expression
vectors for large proteins.
1989 A gene responsible for cystic fibrosis was explored.
1990 Human Genome Project was launched.
1993 Kary Mullis won the Nobel Prize in Chemistry for inventing the
tool of PCR. FDA approved a recombinant protein to treat
multiple sclerosis.
1994 A number of genes, human and others were identified and their
functions explained.
1995 The first vaccine for hepatitis A was explored.
Researchers at the Institute for Genomic Research completed the
first full gene sequence of a living organism for the bacterium
Haemophilus influenzae.

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1997 Researchers at the Roslin Institute in Scotland cloned a sheep

called Dolly from the cell of an adult ewe. Dolly was the first
mammal cloned by a technique called nuclear transfer
technology.
The first human artificial chromosome was discovered.
1998 A group of researchers succeeded in culturing embryonic stem
cells.
A draft of the human genome map is created that locates more
than 30,000 genes.
Embryonic stem cells were employed to regenerate tissue and
produce disorders mimicking diseases.
2000 Sir Hargobind Khorana synthesized DNA in a test tube. He
demonstrated the role of nucleotides in protein synthesis and
helped in finding the genetic code.
2001 The journals Science and Nature reported the human genome
sequence.
2002 A very rapid shotgun sequencing of major genomes was
completed.
2003 Celera and the NIH successfully finished the sequencing of the
human genome.
2004 The FDA supported the first monoclonal antibody for cancer
therapy.
2006 The FDA sanctioned a recombinant vaccine against human
papilloma virus.
Researchers established the three-dimensional (3D) structure of
HIV.
2008 Japanese chemists developed the first DNA molecule made
nearly entirely of artificial parts.
2009 Scientists identified three new genes connected with Alzheimer's
disease, paving the way for possible new diagnostics and
therapeutics.
2013 Researchers established the three-dimensional (3D) structure of
HIV, which causes AIDS.
2010 Researchers from the J.Craig Ventere Institute create the first
synthetic cell.
2011 Trachea derived from stem cells transplanted into human
recipient.
Advances in 3-D printing technology lead to “skin-printing.”

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2012 Synthesis of a polymer- XNA, third information-storing molecule

by molecular biologists Vitor Pinheiro and Philipp Holliger of the
Medical Research Council in the United Kingdom. Just like
DNA, XNA is capable of storing genetic information and then
evolving through natural selection.
2013 Researchers in Japan developed functional human liver tissue
from reprogrammed skin cells.
2014 Created new DNA bases in the lab, expanding life’s genetic code
and opening the door to creating new kinds of microbes.
2015 Researchers in Sweden developed a blood test that can detect
cancer at an early stage from a single drop of blood.
2016 For the first time, bioengineers created a completely 3D-printed
‘heart on a chip.’
2017 Discovered a new molecular mechanism that might be the cause
of severe premenstrual syndrome known as PMDD.
Scientists engineer disease-resistant rice without sacrificing yield.
Blood stem cells grown in lab for the first time.

4.5 TYPES OF BIOTECHNOLOGY

The application of biotechnology has grown in recent years. In accordance with the field in
which it is used, it has been categorized into various types.
1. Agricultural Biotechnology
Biotechnology has been used in developing genetically modified plants to increase crop
production or introduce characteristics that impart tolerance against biotic and abiotic stress,
provide resistance against pest and pathogen attack. The contribution of biotechnology in the
field of agriculture includes organic agriculture, agrochemical based agriculture, and genetically
engineered crop based agriculture etc. Examples- development of crops that express anti-pest
characteristics; the genes of Bacillus thuringiensis have been transferred to crops; a protein (Bt)
produced by Bacillus thuringiensisis found to be very effective against pests such as the
European corn borer.
2. Medical Biotechnology
It deals with the application of living cells and other substances to improve human and
plant health. It is utilized as a research tool for studying human and plant health, identifying
pathogens, and human cell biology, as well as for the treatment of diseases or the prevention of
diseases. There is a huge variety of biotechnology products available for medicinal application,
as outlined in Table 4.2. Some products are designed to resemble their human counterparts,
while others are made to be different from them. These products can be analogues, ones that
have undergone chemical modification (such being pegylated), or they can be completely new

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(e.g., single chain or fragment antibody products, gene transfer vectors, tissue-engineered
products). The procedure is employed to produce pharmaceutical medications and other
compounds that combat diseases. The field is used for development of new drugs and treatments.
An anti-lymphoma vaccination has been created using genetically modified tobacco plants that
exhibit RNA (a chemical similar to DNA) from malignant (actively cancerous) B-cells.
Table 4.2 Biotechnological products in medicine
Class Products
Hormones Growth Hormone, Follicle stimulating hormone, Insulin, Insulin analogues
Growth factors Platelet-derived growth factor, Nerve growth factor, Insulin growth factor-I
Cytokines Interferones, Interleukins, Colony stimulating factor, Erythropoietin
Vaccines Conventional, Recombinant protein antigen, Modified bacteria or viruses
Nucleic acid Gene therapy, DNA vaccines, Ribozymes
based products
Cell, tissue and Autologous, Xenoxenix
organs
Others Clotting factors, Enzymes

3. Industrial Biotechnology
Biotechnology is also used in the industrial sector. It comprises the utilization of microorganisms
or parts of cells, like enzymes, to produce products that are valuable for the industrial sector,
such as food and feed, chemicals, detergents, textiles, paper and pulp, biofuels, and biogas.
Examples: Industrial biotechnology has enabled the development of biocatalysts for the synthesis
of chemicals.
4. Environmental Biotechnology
Environmental biotechnology is a branch of biotechnology that uses biological processes to
address environmental issues like the genetic rescue of a species, the elimination of pollutants,
the manufacture of renewable energy sources, or the production of biomass. Examples: In
bioremediation, the potential of living organisms is used to remove and treat waste. The
development of enzyme bioreactors allows for the efficient removal of industrial and food waste
components via sewage systems. Recently, a new classification scheme for biotechnology was
adopted. Different biotechnology branches have been assigned different colours in this form of
classification (Table 4.3 and Fig. 4.1.).
Table 4.3 Classification of different biotechnology fields based on colour.
Color designation Description

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Gold biotechnology Bioinformatics is connected to gold biotechnology. It can be focused

into two main functions: creation and maintenance of databases of
biological information & designing and manipulating of biological
materials It is related to areas of life science, computer science,
statistics, and specialised biological fields like genomics, proteomics,
waste generation, agriculture, and drug development.
Red Biotechnology Red or medical biotechnology is related to the application of
biological techniques that can be used for the diagnosis, prevention,
and treatment of diseases. It encompasses the creation of novel
pharmaceuticals, vaccines, and antibiotics, as well as regenerative
therapies, molecular diagnostics, and genetic engineering methods to
treat diseases by genetic manipulation.
White Biotechnology White biotechnology is connected to industrial processes. It is a
modern application that uses limited resources than conventional
methods to produce industrial items while utilising living organisms
like yeast, fungi, bacteria, plants, and their enzymatic systems. Such
processes are employed globally in industries such as chemicals,
pharmaceuticals, food and feed, detergents, pulp and paper, textiles,
materials, and polymers, as well as the energy sector. It includes
designing of energy-efficient, less polluting, and low resource-
consuming processes and products.
Yellow Biotechnology Applies to the use of biotechnology in the production of food, such as
the fermentation of wine, cheese, and beer. The main purpose of
yellow biotechnology is improvement of certain food to obtain the
most nourishing one.
Grey Biotechnology Environmental applications are connected to grey or environmental
biotechnology. It can be separated into two categories: preserving
biodiversity and using microbes to remove contaminants
(bioremediation). Through the use of biological systems in waste
treatment and management, as well as for the protection and
restoration of the environment, grey biotechnology plays a critical
role in resolving environmental issues.
Green Biotechnology It is connected to the agricultural process with the goal of enhancing
nutrient quality, producing superior disease-resistant plants,
increasing productivity, and lowering production costs. Genetic
alteration is used in this area to produce transgenics. The
development of genetically modified organisms, the production of
synthetic seeds for commercial use, and tissue culturing and
micropropagation are some of this field's major applications.
Blue Biotechnology Blue or marine biotechnology is based on the use of marine and

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aquatic organisms for requirement of new substances that are used in

various industrial sectors and also for the conservation of the
environment. It deals with the law, ethical and philosophical issues
related to biotechnology.
Violet Biotechnology Aspects of law, ethics, and philosophy are connected to violet
biotechnology. Significant advancements in biotechnology have been
made in a number of fields, including the industrial, agricultural, and
medical sectors. There are also some religious, moral, and societal
difficulties in this area.
Brown biotechnology Brown biotechnology is related to management of desert and dry
regions which makes larger part of the earth. Main purpose of this
arid zone biotechnology is improved seeds and high-quality disease-
free plants.
Dark biotechnology It is connected to bioterrorism and biological weapons. This industry
produces a large variety of disease-causing biological agents with
minimal production costs and simple transportation from one location
to another. In this field, germs and toxins, particularly those of plant
and animal origin, are employed as raw materials to cause disease and
death in people, crops, and other living things.

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Figure 4.1: Classification of biotechnology based on color. (Source: Padhy et al., 2020)

4.6 APPLICATIONS OF
BIOTECHNOLOGY Biotechnology's role in combating the
Covid 19 pandemic
Biotechnology has been successfully used in
many fields, including the environment, In the campaign against COVID-19, biotechnology has
health care, agriculture, business, and many taken the lead. Using biotechnological methods,
more. The applications of biotechnology in vaccines have been manufactured to treat corona
different areas are given in table 4.4 virus infections. For COVID-19, Pfizer-BioNTech and
Moderna created high-efficiency mRNA vaccines. It
1. Agriculture
has been demonstrated that the mRNA vaccine
The Green Revolution began as a result of created by BioNTech/Pfizer is 90% effective in
the introduction of biotechnology in preventing COVID-19 infection. Other vaccines have
agriculture. Organic farming, agrochemical- also been created using other methods, including
based farming, and genetically modified interference RNA that stops the virus in nebulizers,
crop-based farming are few examples of recombinant ACE2 proteins that deceive the virus
how biotechnology has benefited into not binding to human cells, and antibodies that
agriculture. The introduction of pest- fight the virus. Astra Zeneca developed the Sputnik V
resistant plants and genetically modified and AZD1222 viral vector vaccines. Moderna
crops has helped to enhance food production (Cambridge, MA, and USA) and Pfizer developed the
and fulfil the needs of the growing human mRNA vaccines mRNA-1273 and BNT162b2 (New
population. It has also helped to triple food York, USA)
production.
The development of crops with higher yields, improved nutritional value, and resistance to a
variety of biotic and abiotic challenges has proven to be a very fruitful use of biotechnological
approaches. With the help of genetic engineering, food, horticultural crops, and tree species have
all been successfully altered. One example is the modification of oil seeds to produce fatty acids
for petrochemicals and fuels. There have been initiatives to create plant-based vaccinations.
There have been produced crops that exhibit tolerance to a wide variety of insects and pests.
2. Medicine
The development of vaccinations, medicines, diagnostics, and human genome research has all
made use of biotechnological methods. One of the main uses of biotechnology in medicine is the
production of insulin and vaccines like the hepatitis B. By allowing the mass production of
effective therapeutics, recombinant DNA technology has made a significant impact on the
healthcare industry. Transgenic animals have been used to study how genes influence the onset
of human diseases such cancer, cystic fibrosis, rheumatoid arthritis, and Alzheimer's. For
instance, genetically engineered insulin called Humulin is used to treat diabetes. The insertion of
genes into a person's cells and tissues during gene therapy has made the treatment of genetic

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disorders feasible. PCR and ELISA are two further examples of how biotechnology is used in
medicine.
3. Environment
Environmental issues have been identified, monitored, and remedied using biotechnological
approaches. Waste management, bioremediation (the use of live organisms to treat
environmental toxins), and phytoremediation method (use of plants to remove pollutants from
the environment) have all benefited from the application of this. In addition to these renewable
resources, biotechnology has also been used to produce biodegradable goods and alternative
energy sources. By removing a wide range of pollutants from diverse areas of the environment,
biotechnology is used to produce bacteria with tremendous potential for environmental cleanup.
4. Industries
For a variety of purposes, including the development of new materials for the construction
industry, the production of cellular structures or the production of biological components,
biotechnology has been used. Other applications include the production of beer and wine,
washing detergents, and personal care products. For the finishing of fabrics and apparel,
biotechnology is employed in the textile industry. Along with additional qualities like greater dye
uptake and retention, increased absorbency, and wrinkle- and shrink-resistant fabric, it aids in the
manufacturing of warmer, stronger, warmer, and wrinkle- and shrink-resistant clothing. In the
food processing business, biotechnology has been successfully implemented.

5. Biofuels
In the area of energy production, biotechnology has been applied. Biofuel can be made from the
waste from bioremediation and used to power generators. Methane generation may be aided by
bacteria that break down sulphur liquor, a waste product of the paper manufacturing sector.
6. Evolutionary studies
Genes associated with ecological traits and evolutionary diversification can easily be traced
using biotechnological tools.
7. Other Uses
• Biotechnology has used fermentation for centuries. Microorganisms like yeast have long
been used in the production of alcohol and bread. In the current scenario, cultures have
been purified and genetically improved to generate superior quality food items.
• Crop enhancement through the breeding of plants with desirable features is another way
that biotechnology is used in agriculture.
• Transgenic plants are created through genetic engineering to have certain traits.

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• Tissue culture is another application of biotechnology to produce a large number of

plants with an explants. It also helps in increasing the number of endangered plant
species.
• Enzymes needed to convert plant and vegetable sources into the components of
biodegradable polymers can be produced by microbes under the control of certain
conditions.
• It is also helpful in forensics for the identification of criminals, or in paternal disputes.

Table 4.4 The applications of biotechnology in different areas.

Area Applications
Plant biotechnology Transgenic plants, production of secondary metabolite, production of
pathogen-free plants or crop improvement, production of herbicide-
resistant crops, pest-resistant ('Bt concept' pest-resistant transgenic)
plants, drought resistance, flood resistance, salt tolerance, high-yielding
GM crops, nitrogen fixing ability, acidity and salinity tolerance, in-vitro
germplasm conservation, genetic variability, in-vitro pollination,
induction of haploidy, somatic hybridization, genetic transformation,
somatic embryogenesis, organogenesis, phytoremediation, in-vitro plant
germplasm conservation, mutant selection, somaclonal variation, plant
genome analysis, hybrid seeds, artificial seeds.

Animal biotechnology Biopharmaceuticals: Production of hormones, growth factors, interferons,

enzymes, recombinant proteins, vaccines, blood components,
oligonucleotides, transcription factor-based drugs oligonucleotides.

Antibiotics, Diagnostics: antibodies, biosensors, PCR, therapeutics,

vaccines, medical research tools, human genome research, development
of biosensors.

Gene therapy, Stem cell therapy, Animal tissue culture: Cell, tissue and
organ culture, Gene cloning: rDNA technology, genetic engineering,
transgenic animals, antibiotics, DNA markers, animal husbandry,
xenotransplantation, medical biotechnology.

Biopharmaceuticals (drug or vaccine developed through biotechnology),

therapeutants, i.e. products used to maintain health or prevent disease,
biopharming, i.e. production of pharmaceuticals in cultured organisms.

Biopolymers, Designer drugs.

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Agricultural crop biotechnology, horticultural biotechnology, tree biotechnology, food

biotechnology processing, plant biotechnology (photosynthesis improvers, bio-fertilizers,
stress-resistant crops and plants, bio-insecticides and biopesticides)

Food: Increased milk production

Pharmaceuticals: Animals engineered to produce human proteins for

drugs, including insulin and vaccines

Industrial Metabolite production (antibiotics, acetone, alcohol, enzymes, vitamins,

biotechnology organic acids), anaerobic digestion (for methane production), waste
treatment (both organic and industrial), production of bio-control agents,
fermentation of food products, bio-based fuel and energy, recovery of
metals and minerals, bioethanol, pulp and paper, food, textiles and
leather, pharmaceuticals, an enzymatic process for producing antibiotics.

Environmental Environmental monitoring, Waste management, Pollution prevention.

biotechnology
Fuel and fodder Tissue culture technique for rapid afforestation of degraded forests,
Renewable fuels and regeneration of green cover

Aquatic Aquaculture, environmental remediation, marine byproducts for human

health, biomaterial and bioprocessing, restoring and protecting marine
biotechnology
ecosystems, improving seafood quality, marine molecular biotechnology.

4.7 VARIOUS TECHNIQUES IN BIOTECHNOLOGY

In biotechnology, a wide range of techniques have been used. Table 4.5 lists some of the most
significant ones.

Table 4.5 Various techniques used in biotechnology.

Techniques Description

Genetic Engineering The procedure involves altering an organism's DNA by introducing

Technology foreign DNA.

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Enzyme engineering With this method, an enzyme's effectiveness can be increased or its
activity can be generated by changing the amino acid sequence.
Genetic engineering techniques are widely used to improve enzyme
efficiency.

Protein Engineering The process involves altering current proteins or developing new
Technology proteins to produce useful products.

Cell and Tissue Culture In this technique, cells/tissues are grown under laboratory conditions to
Technology produce a organism or new products.

Bioinformatics Technology Computational analysis of biological data, e.g., sequence analysis

macromolecular structures, high-throughput profiling data analysis.

Antisense or RNAi A technique known as RNA interference (RNAi) controls the

Technology expression of genes in eukaryotic organisms by using short RNAs
(RNAs with less than 30 bases). Research on gene function and
therapies for the treatment of disease have both benefited from an
RNAi-based method.

Protein separation and Procedures for separating and identifying desired DNA, including
identification techniques electrophoresis, microarrays, and blotting.

Genomics The genome approaches are used to determine the biological function
of genes and their products. Transcriptomics (profiling of microarray
expression), proteomics (structures/ modifications/ interactions of
proteins), metabolomics (e.g. metabolite profiling, chemical
fingerprinting, flux analysis) are some of its diversifications

4.8 PLANT BIOTECHNOLOGY

New plant traits and varieties can be developed with the help of plant biotechnology. Plant
biotechnology refers to procedures or scientific methods used to produce useful plants, their
products, and the development of novel plant features. Utilizing biotechnological technologies
accelerates crop improvement and makes it easier to transfer genes from unrelated species.
The two processes involved in plant biotechnology are tissue culture and genetic engineering.
The most common approach in plant biotechnology is plant tissue culture. In 1939, Gautheret att
empted to grow isolated cells and root tips on a structured medium, which was the beginning of p
lant tissue culture. The tissue culture techniques provide the platform for the rapid multiplication

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of plant species. The majority of plant tissue culture enterprises take advantage of the basic
ability of plant cells to regenerate in order to produce elite varieties with superior genotypes
quickly and on a big scale in a comparatively short time frame. The plants are genetically
modified plants by transferring genes from one organism to plant to get the desired
characteristics. Plants have been genetically modified to induce herbicide tolerance, salinity
tolerance, drought tolerance, pest resistance, enhanced nitrogen-fixing ability, improved
nutritional value and food quality.
Today's plant biotechnology represents a new era in science and technology, one that prioritizes
the production of secondary metabolites, significant genetic advancements for plants, the
conservation of germplasm, and the mass production of disease-free and novel kinds. Production
of artificial seeds, biopharmaceuticals, plant-made pharmaceuticals, recombinant or other
therapeutic proteins, transgenic plants, and plant-made vaccines or antibodies is part of the
modern research work in plant tissue culture science.

4.9 METHODS IN PLANT BIOTECHNOLOGY

The following techniques are used in plant biotechnology.
• Plant tissue culture
• Genetic engineering/ recombinant DNA technology
• Molecular Assisted Breeding

4.9.1 Plant Tissue Culture

The plants are raised in a laboratory environment using cells, anthers, pollen grains, or other
tissues. Then they are multiplied by a huge number. Explants from plants or tissues that produce
calluses (undifferentiated masses of cells) can directly give rise to somatic embryos under certain
cultural settings. Due to the rapid plant multiplication facilitated by plant tissue culture, breeders
are able to introduce new cultivars considerably more quickly.
The steps in the tissue culture procedure include placing the tissue on a nutrient medium,
allowing the cells to multiply to form a callus or the embryos, moving them to a medium
containing plant growth regulators, and then allowing the embryos to produce roots (Fig. 10.6).
The seedlings are moved into greenhouses. Through the use of plant tissue culture, a wide range
of food and medicinal plants have been cultivated. Cell culture enables the production of single-
cell clones, embryo rescue, the introduction of traits like disease resistance into elite breeding
lines, as well as the generation of haploid plants allowing the development of homozygous
breeding lines. This technique is now widely used for the improvement of important crops
including major cereals, legumes and trees.

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Figure 4.2: Sketch out of plant tissue culture technique (Source: sanjeetbiotech.blogspot).

4.9.2 Genetic engineering

Genetic engineering is the process of changing an organism's genetic (hereditary) material. The
removal of a gene or genes from one organism and their transfer to another organism is known as
genetic engineering or recombinant DNA technology. A transgenic plant or genetically modified
organism is produced when additional DNA is incorporated into the native DNA of a plant
(GMO). Restriction enzymes, cloning carriers (vectors) to carry the desired genes, and detection
and selection of cloned genes are the fundamental elements for successful genetic engineering.
In genetic engineering, various gene transfer strategies are employed, such as:
• Agrobacterium-mediated gene transfer: The desired trait is extracted from an organism's
DNA, inserted to an Agrobacterium, and then transferred to the target plant. The DNA is
accepted by cells, which develop into plants with the new trait.
• Gene gun: DNA that codes for the desired trait is coated onto tiny particles of tungsten
and fired into a group of plant cells. The desired feature is displayed in cells that receive
the DNA.
• Polyethylene glycol (PEG) is a chemical that enables the quick uptake of DNA by plant
protoplasts and integration into the chromosomal DNA of the plant.
• Another method is electroporation, in which short, high-voltage electrical pulses cause
the formation of transient micropores in cell membranes, allowing DNA to enter the cell
and then the nucleus.
• In addition to these, other techniques for transferring DNA include lipofection,
microinjection, and calcium phosphate transfection.
Agrobacterium vectors are commonly used for the transfer of transgenes into plants The DNA
sequences may be cut at various points, and the resultant fragments may then be placed into
various locations throughout the genome (Fig. 4.3). Transgenes inserted into the genome contain
regulatory elements and a selectable marker, often an antibiotic‐ or herbicide‐resistant gene.

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Figure 4.3: Diagrammatic representation of plant genetic engineering (Source: Kumar & Prasad, 2019)

4.9.3 Molecular Assisted Breeding (MAB)/Marker Assisted Selection (MAS)

Marker-assisted breeding, commonly referred to as molecular breeding, is a modern breeding
method where the selection of desired traits is based on particular molecular markers such as
simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) or molecular
breeding is the process of selecting qualities by using DNA markers that are closely linked to
phenotypic traits. Genetic markers such as microsatellites, random amplified polymorphic DNA
(RAPD), restriction fragment length polymorphisms (RFLPs), amplified fragment length
polymorphism (AFLP) are important components of molecular breeding. Plant genetic
improvement is facilitated by the data produced by molecular markers, transcriptome profiling,
and genome sequencing. Molecular markers have been developed for many crops including
trees. The markers can be used to track the presence of valuable characters in large segregating
populations as part of a crop-breeding program
A new molecular breeding technique for crop development is genomic selection. It helps to
segregate breeding populations and identify superior genotypes with higher breeding values (Fig.
4.4). It is based on genome-wide marker profile data. MAS have proved as an efficient breeding
method for drought and salt-tolerant oilseed crops such as Brassica. It has provided valuable
contributions in the designing and development of oilseed crop ideotype(s).

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Figure 4.4: Marker-assisted breeding method process (Source: Collard Bertrand & Mackill David, 2008)

4.10 APPLICATIONS OF PLANT BIOTECHNOLOGY

Plant biotechnology has several, wide-ranging applications. Basic research and industrial
applications are some of these. Understanding ideas like molecular pathways in plant cells is the
fundamental application of plant biotechnology. Plant biotechnology techniques such as tissue
culture and genetic engineering have been applied to plants for modification and improvement.
These include growing pathogen-free plants, conserving genetic material, clonal propagation,
producing secondary metabolites, and studying cell behavior. Biotechnology has got its
application in crop improvement programs. There are many areas where plant biotechnology is
used, including:
1. Improvement of varieties for various agronomic traits such as:
• Crop improvement (resistance to biotic stress: pests, viruses, pathogens; abiotic
stress: tolerance to drought, salinity).
• Increasing the shelf life to delay the fruit ripening.

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• Value addition: enrichment of crops with vitamins.

• Ornamental plants for characters such as size, color, smell
• Food processing.
2. Phytoremediation: removal of contaminants.
3. Biofuels: bioenergy crops.
4. Commercial products such as biopolymers, therapeutic proteins, biodegradable plastics, etc.
5. Protection of natural biodiversity.

4.10.1 Crop Improvement

Plant tissue culture technique
Plant clonal multiplication and propagation are facilitated by tissue culture. This is called
micropropagation. The offspring produced by vegetative propagation i.e. asexual reproduction of
a single plant or individual is called a clone. To develop plantlets, shoot buds produced on
explants are separated and rooted. The technique proves useful in eliminating viruses and other
pathogens. Useful chemical compounds are produced on a big scale using plant cell suspension
culture and immobilized cells. The preservation of vegetative tissues and the conservation of
endangered genotypes are both facilitated by cell and organ culture. The plants that do not
produce seeds (sterile plants) or have ‘recalcitrant’ seeds can be preserved via in vitro
techniques. Cryopreservation involves the storage of cells or tissues in liquid nitrogen at an
extremely low temperature (−196°C) under in vitro conditions. This technique helps in the long-
term conservation of important biological material and genetic resources. The embryonic tissues
are generally cryopreserved for future use.
4.10.2 Genetic Transformation
Transgenic plants are produced using the genetic transformation approach by transferring the
desired trait's genes into the host plants. In plants, genetic transformation is completed by vector-
mediated i.e. Agrobacterium-mediated genetic transformation, or vector less i.e. direct gene
transfer methods. The method presents a significant opportunity for genetic improvement in
different crop species. Plant biotechnology and breeding programmes are integrated using this
method. Plants can be genetically modified to have traits like higher yield and better quality. For
instance: The development of transgenic "golden rice." Transgenic rice contains three genes
encoding the enzymes responsible for the conversion of geranylgeranyl diphosphate to β-
carotene (provitamin A). The transgenic rice produced is enrich in iron content. The plants are
obtained by expressing ferritin or metallothionein transgenes, or making the existing iron more
available by reducing levels of the iron-sequestering protein, phytase.
Transgenes can be used to create cells with a high concentration of the desired substances, which
can have industrial applications. The modification of exogenous compounds by plant cells is
called biotransformation. The enzymes found in the plant cell catalyze the bioconversion

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reactions. Plants that exhibit resistance to or protection against numerous pests, illnesses, and
viruses have been developed using the genetic engineering technique.
4.10.3 Pharmaceuticals
Using both conventional and novel methods, including genetic engineering and plant cell culture,
it is possible to create medicines, secondary metabolites, proteins, medications, and vaccines
from plants.
In addition of this, a new branch of biotechnology i.e. nanobiotechnology which involves the use
of nanomaterials has proven useful in the development of crops mainly by manipulating the plant
nutrient content, imparting disease and pesticide resistance in plants. In order to create nano
insecticides, metallic nanoparticles with comparatively superior anti-pathogenic, anti-fungal, and
anti-bacterial properties are utilised. Iron and zinc oxide nanoparticles function as nanofertilizers
by facilitating the plant's efficient uptake of nutrients. Their effectiveness is dependent on a
variety of variables, including plant susceptibility, size, content, and chemical characteristics of
nanomaterials.
4.10.4 Industrial
Plants produce a variety of products that are used commercially, including proteins, antibodies
(plantibodies), and fuels. For instance avidin and β-glucuronidase (GUS) are proteins produced
in transgenic maize. Avidin is utilised as a biopesticide as well as a biochemical reagent for
research and diagnostics. Oleochemicals are made from plant oils, which are employed as a
feedstock. The soybean crop has been transformed to get high oleic acid content.
Starch produced by plants has also got uses in industry. Interest is also developed in
manipulating complex carbohydrates in cereal crops such as rice, wheat, maize and barley. The
starches are used for the development of packaging materials or even biodegradable plastics.
Many of the enzymes involved in starch biosynthesis have been characterized and their genes
cloned and attempts are been made to develop plants with enhanced capacity for the production
of starch.
Polyhydroxyalkanoates (PHAs), which are non-toxic biodegradable polymers made by plants,
can be produced more abundantly through agricultural practices. The polymer can be extracted in
large quantities from seeds or leaves. To increase the production of these copolymers in the
plant, efforts are being made to acquire transgenic plants (including one in oil palm).

4.11 SCOPE OF BIOTECHNOLOGY

The field of biotechnology is enormous in and of itself, and it encompasses many areas of
biology. This comprises plant tissue culture, production of transgenic in animal and plants,
development of monoclonal antibodies, uses in medicine as tools and therapeutics, production of
new enzymes and their immobilization for industrial use, and control of pollutions, etc.

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As we can say the 20th century was the era of electronics, similarly 21st century can be
designated as the era of biotechnology. Biotechnology has rapidly become a field of activities
with notable effects on all facets of human welfare, including food processing, environmental
protection, human health, and overall quality of life. Biotechnology is making significant
contributions in a number of fields, such as environment, medicine, agriculture, horticulture,
floriculture, dairy, forestry, human health, animal health, fisheries, aquaculture, food processing,
mining, animal husbandry, renewable energy, crime detection, parental dispute etc.
Fundamentally, the goal of biotechnology is to enhance human life quality and safeguard against
harmful diseases. The scope of biotechnology can be summarized below:
1. Developing immobilized cell and enzyme systems for chemical process industries.
2. For increasing disease-resistant, high-yielding varieties of crops,
3. Production of biocides in agriculture,
4. To introduce harmless bio fertilizers instead of harmful chemical fertilizers,
5. Genetical improvement of microorganisms for production of pharmaceutical products.
6. To preserve germplasm of plants, animals and microbes,
7. To develop automated bio-screening for therapeutic agents
8. To produce pharmaceutical products to treat severe diseases in man and animals,
9. Production of Bio-processing alkenes to valuable oxides and glycols
10. To produce biofuels for reducing the felling of forest trees for fuel wood,
11. Engineering of a series of organisms for specific industrial use.
12. To make use of various microorganisms in food making and preservation of food,
13. To employ microorganisms in the extraction of minerals from their poor quality ores,
14. To minimize pollution hazards.
15. In Human gene therapy.
16. For improved production of Vitamin B12.
17. Manufacturing ethanol by continuous fermentation.
18. Microbiological based production of human insulin and interferon’s,
19. Production of monoclonal antibodies for organ transplant tissue typing.
20. Production of photo-synthetically efficient plants.
21. Production of transgenic plants and animals.
22. Production of xanthan gum in oil fields for recovery of crude mineral oils.

4.12 SUMMARY
The use of living organisms to create technologies and goods for human improvement and
sustainable development is known as biotechnology.
Biotechnology is the use of living systems and organisms to produce or develop useful products.
It can also refer to "any technological" application that uses biological systems, live organisms,

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or their derivatives to produce, alter, or produce goods for a specific purpose. The application of
biotechnology in agriculture, food production, and medicine has been practiced by humans for
thousands of years.
The term "Biotechnology" was first used by Hungarian engineer Karl Ereky in 1919. He defines
biotechnology as the use of living organisms to the production of valuable resources or products.
In other words, it is the technology that uses biological mechanisms, systems, or processes to
produce products that enhance human life.
Modern biotechnology has got its applications in areas such as medicine, healthcare,
therapeutics, diagnostics for development and production of novel drugs, agriculture for
development of improved crop varieties, environment for treatment of waste and prevents
pollution.
Plant biotechnology is referred to the use of plant cells, tissues, organs for the generation of
useful products. The plant parts (explants) are cultured under in vitro conditions to get plantlets.
Plant biotechnology plays a promising role in the field of agriculture, industry and
pharmaceutical sciences.
Plant biotechnology has many applications such as large scale production of biochemicals, rapid
clonal multiplication, development of homozygous lines via production of haploids, conservation
of germplasm etc. The main methods in plant biotechnology are plant tissue culture, genetic
engineering and marker assisted breeding. Clonal multiplication, raising of virus-free plants can
be done via plant tissue culture. The development of plants with tolerance against abiotic and
biotic stresses, better nutritional quality, and better storage can be achieved via manipulation of
DNA by genetic engineering.
Biotechnology is making significant contributions in a number of fields, such as environment,
medicine, agriculture, horticulture, floriculture, dairy, forestry, human health, animal health,
fisheries, aquaculture, food processing, mining, animal husbandry, renewable energy, crime
detection, parental dispute etc. Fundamentally, the goal of biotechnology is to enhance human
life quality and safeguard against harmful diseases.

4.13 SELF ASSESSMENT QUESTIONS

4.13.1 Multiple Choice Questions
1. Who coined the term ‘biotechnology’?
a) Karl Ereky b) Arthur Kornberg
c) Both a & b d) Chaim Weizman
2. -------------------is a product of biotechnology.
a) Skin b) Virus
c) Vaccine d) Fungi

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3. The Golden Rice variety is rich in:

a) Biotin b) β-carotene and Ferritin
c) Vitamin C d) Thiamine
4. In which the following industrial areas, biotechnology is applicable?
a) Environment b) Health care
c) Agriculture d) All of the above
5. Tissue culture technique has been biotechnologically successful in production of
a) Beverages b) Plantlet
c) Insulin d) Cheese
6. Transgenic plants are produced by using Ti plasmid from the
a) E. coli b) Agrobacterium tumefaciens
c) Bacteriophage d) Bacillus thuringiensis
7. The biotechnology has contributed to field of
a) Pharmacy b) Industry
c) Agriculture d) All above
8. Bt stands for
a) Biotechnology b) Bacillus thuringiensis
c) Bollworm toxin d) None of the above
9. Which of the following techniques use genes and DNA molecules for diagnoses of diseases?
a) PCR b) Recombinant gene technology
c) Gene therapy d) All of the above
10. GMO stands for
a) Genetically modern organism
b) Genetically transferred organism
c) Genetically modified organism
d) Genetically mutant organism
11. What is the ‘part’ of plant used for culture called?
a) Explant b) Callus
c) Both a & b d) None of the above
12. Name the technique used for computational analysis of biologic data.
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a) Python b) DNA fingerprinting

c) Bioinformatics d) SPSS
13. Name the antibiotic produced by Alexander Fleming.
a) Cephalexin b) Azithromycin
c) Amoxicillin d) Penicillin
14. Name of the sheep cloned by researchers in Scotland
a) Dolly b) Morag
c) Megan d) Both b & c
15. Grey field of biotechnology related to:
a) Environmental biotechnology b) Agriculture biotechnology
c) Industrial biotechnology d) Medical biotechnology

4.13.2 Fill in the Blanks

a) Biotechnology is the use of ……………………. to produce or develop useful
products.
b) The plants formed after incorporation of genes for desired characters are called
………………….. .
c) Undifferentiated mass of cells produced via tissue culture is called ……………….
d) Genetically engineered insulin called ………………. is used to treat diabetes.
e) Polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) are
examples of ……………………..
Answer key
4.14.1: 1-(a); 2-(c); 3-(b); 4-(d); 5-(b); 6-(b); 7-(d); 8-(b); 9-(d); 10-(c); 11-(a); 12-(c); 13-(d); 14-
(a); 15-(a)
4.14.2: a) living systems and organisms; b) transgenic; c) callus; d) Humulin; e) genetic markers

4.14 REFERENCES
Campbell, C. S. Biotechnology and the fear of Frankenstein, (2003), Camb. Q. Healthc. Ethics
12 342–52.
Chandrashekara, K. N. and Yakkaldevi, A. “Basic Concept of Biotechnology”, (2015), Laxmi
Book Publication, Solapur.
Collard Bertrand C.Y and Mackill David J. Marker-assisted selection: an approach for precision
plant breeding in the twenty-first century (2008), Phil. Trans. R. Soc. B363557–572.

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Gupta, R. and Rajpal, T. Concise Notes in Biotechnology (Tata/McGraw-Hill Education) ch 1

(2012).
Kumar, A. and Prasad, M. N. V. Plant Genetic Engineering Approach for the Pb and Zn
Remediation: Defense Reactions and Detoxification Mechanisms. In: Transgenic Plant
Technology for Remediation of Toxic Metals and Metalloids (2019), Pages 359-380.
https://doi.org/10.1016/C2017-0-01241-7.
Padhy, I., Mahapatra, A. P. K., Saraswat, R. and Song, J. Role of biotechnology in
pharmaceutical research: A comprehensive review, (2020), Indo Am. J. P. Sci, 2020;
07(05).
Purohit, S.S. “Biotechnology Fundamentals and Applications”, Third Edition (2004), Agrobios
India.
Samiksha, S. Biotechnology: meaning, technologies and applications in India (2017),
www.yourarti clelibrary.com/biotechnology/biotechnology-meaning-technologies-and-
applications-in-india8617-words/11249 (Accessed 1 July 2017).
Srivastava, H.N. “Plant Physiology, Biochemistry and Biotechnology”,Tenth Edition (2013),
Pradeep Publication Jalandhar.
http://sanjeetbiotech.blogspot.com/2013/03/plant-tissue-culture-biotechnological.html
Verma, A. S., Agrahari, S., Rastogi, S. and Singh, A. Biotechnology in the realm of history.
(2011), J. Pharm. Bioallied Sci. 3 321–3.

4.15 SUGGESTED READINGS

Chawla, H. S., Introduction To Plant Biotechnology (2003), Oxford & Ibh, Second edition .
Dubey, R.C., Text book of Biotechnology (1993), S. Chand & Company Ltd., Ramnagar, New
Delhi.
Anita Dua, A., and Mahajan, R. Introduction to Basic of Biotechnology, First edition (2012),
Vayu Education of India.
Gupta, P.K. “Elements of Biotechnology”, First Edition (1999), Rastogi Publications, Meerut,
U.P.
ICAR eCourse, Principles of Plant Biotechnology, https://pravara.in/wp-
content/themes/twentyseventeen/essentials/pdf/elearn/Principles-of-Plant-Biotechnology.

4.16 TERMINAL QUESTIONS

4.17.1 Short Answer Type Questions
1. What is the basic concept of Biotechnology?

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2. What do you understand about green biotechnology?

3. What do you mean by white biotechnology?
4. What is the use of biotechnology in the field of medicine?
5. Write a short note on Genetic engineering
6. How does Plant Biotechnology play a role in germplasm conservation?
4.17.2 Long Answer Type Questions
1. Discuss various techniques of Biotechnology.
2. Describe in brief various applications of Biotechnology.
3. How does Biotechnology be used for the improvement of crop plants?
4. Describe the methods of Plant Biotechnology.
5. Enumerate various applications of Plant Biotechnology.
6. Give an account on various stages of biotechnology development.

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UNIT 5: PLANT CELL, TISSUE AND ORGAN CULTURE

AND APPLICATION OF PLANT TISSUE CULTURE

Contents:
5.1 Objective
5.2 Introduction
5.3 Plant cell, tissue and organ culture
5.4 Totipotency and Concept of cellular differentiation
5.5 Application of Plant Tissue culture
5.6 Clonal Propagation
5.7 Artificial seed
5.8 Production of hybrids and somaclones
5.9 Production of secondary metabolites/ natural products
5.10 Cryopreservation and germplasm preservation
5.11 Summary
5.12 Glossary
5.13 Self Assessment Question
5.14 Reference
5.15 Suggested Readings
5.16 Terminal Questions

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5.1. OBJECTIVES
After reading this unit you will be able to
• Describe plant cell, tissue and organ culture.
• Understand the concept of cellular differentiation.
• Know about various application of plant tissue culture.
• Understand about clonal propagation and artificial seed.
• Describe the production of hybrids, somaclones and secondary metabolites.
• Know about the process of Cryopreservation and germplasm preservation.

5.2. INTRODUCTION
You have already learned in the previous unit (unit 4) about biotechnology, basic concepts, its
principle and scope. Through previous unit you must have understood that biotechnology
involves the technology that utilizes biological system to obtain useful products for mankind.
One of the important aspects of biotechnology is tissue culture techniques that have led to the
novel possibilities in the field of agriculture, horticulture, forestry, medicines etc. In this unit we
will discuss about plant cell, tissue and organ culture and its history and scope. We will also
discuss the concept of totipotency, cellular differentiation and application of plant tissue culture
in agriculture, forestry, medicines and industry. A study of this unit will help you to understand
the concepts of clonal propagation, artificial seed, somatic hybrids, somaclones, secondary
metabolites production and cryopreservation etc.

5.3. PLANT CELL, TISSUE AND ORGAN CULTURE

Plant tissue culture is a collection of various techniques used in maintaining and growing plant
cells, tissues or organs under sterile conditions on a nutrient medium. Plant cell, tissue and organ
culture have developed very rapidly in past few decades and it has been proved a major
biotechnological tool in the field of agriculture, forestry, horticulture etc. Through techniques
like micropropagation/ clonal propagation, artificial seeds, somaclonal variation etc., many
problems now can be solved which were not feasible through traditional techniques.
The introduction of cell and tissue culture is linked to the discovery of cell and propounding of
cell theory by Schleiden and Schwann (1839). However, Henri-Louis Duhamel du Monceau
(1700-1782) executed the experiments on wound healing in plants through callus formation on
decorticated region of elm plants (Gautheret, 1985). Similarly, Trecul (1853) observed callus
formation in number of plants and Vochting (1878) suggested the presence of polarity as a key
feature guiding the development of plant fragments (Gautheret, 1985). In 1898 and 1902, a
German botanist, Gottlieb Haberlandt done intensive effort and developed the concept of in vitro
cell culture (Krikorian & Berquam, 1969). His experiment failed to induce cell division as till
that time auxin was not discovered. But he lent a foundation to plant physiology. Haberlandt is
thus regarded as father of tissue culture. During 19th century, the concept of callus culture from
different plant parts came into existence and many attempts were made for organ culture. During

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1940-1970, suitable nutrient media were developed for culture of plant cells, tissues, protoplasts,
anthers and embryos. In 1950s, through several important achievements in the field of plant
physiology the understanding of plant growth hormone was increased and this helped in rapid
multiplication of totipotent cell during culture process. In 1960, G. M. Morel laid the foundation
of commercial plant tissue culture through the million fold increase in clonal multiplication of an
orchid (Cymbidium). F. Skoog with C. O. Miller (1957) found out the role of cytokinins in tissue
culture and showed that the ratio of auxin and cytokinin in the growth medium influences the
morphogenesis of shoots and roots during tissue culture. In 1960s, with the identification of the
role of enzymes (cellulase and pectinase) in dissolution of cell wall at suitable pH, isolation and
culture of protoplast was developed. In India, tissue culture work was started during mid 1950s
and was pioneered by Panchanan Maheshwari. Sipra Guha Mukherjeee and S. C. Maheshwari
(1964-67), first time developed the haploid through anther and pollen culture and this discovery
was a landmark in the development of plant tissue culture.

Basic techniques and requirements of Plant Tissue Culture:

The important aspects of plant tissue culture are nutrient medium, maintenance of aseptic
conditions and aeration of the tissue. Plants can synthesize their own food in nature through
photosynthesis but while growing in vitro condition they are heterotrophic and require all
essential minerals, carbohydrate, growth regulators etc. For this, several media have been
developed time to time. Important components of media for plant tissue cultures are inorganic
salts of major and minor elements, iron and carbon source, vitamins and plant growth regulators.
Murashige and Skoog (1962) formulated MS medium suitable for many applications and most
widely used in plant tissue culture. Other media used in plant tissue culture are proposed by
Gamborg et al (1968), White (1963), Heller (1953) and Smith (1967).
The general techniques used in plant tissue culture are cleaning of glassware, sterilization of
glassware and media, sterilization of plant material and culture. For cleaning glassware used in
culture process, it is boiled for two hours in 10% sodium carbonate then after rinsing with tap
water it is soaked in 30% nitric acid overnight. After this it is rinsed with distill water and then
drained, dried and stored in clean place. Nowadays disposable sterile culture vessels are also
available commercially. The cleaned glassware is sterilized using an autoclave or an oven.
Sterilization of culture media involves autoclaving and filter sterilization. Culture media kept in
glass containers sealed with cotton plugs or plastic closures are autoclaved at 15 psi and 121oC
for 15-40 min (exposure time depends upon the volume of nutrient medium). Solution containing
thermolabile compounds like vitamins, plant extracts, amino acids, hormones etc. requires filter
sterilization where these solutions are sterilized using millipore or seitz filter paper (0.2 µm
pore). Surface sterilization of plant material requires greater care as microbes on surface have to
be killed without killing plant tissues. Firstly, plant material is washed under running water and
then treated with detergent solution to remove oils and dirt from the surface. After this, explant is
treated with 70% alcohol for about 1 minute and then washed thoroughly with distill water. The
chemical sterilizing agent mainly used is 20% chlorine water, 1.0% bleaching powder, 0.01% or
0.1% mercuric chloride. After this, the entire sterilized setup i.e. flask containing explants in

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sterilizing solution is transferred to sterile laminar airflow cabinet. Here explants are transferred
to sterile petridish with the help of sterile forceps and then specific portion of explants is
inoculated on sterilized medium. The isolated single cell from cell suspension cultures is capable
of division. They could proliferate and divide to form callus which in appropriate nutrient and
hormonal conditions could regenerate into new plants.

Types of Plant Tissue Culture:

The plant tissue cultures are generally classified on the basis of the type of in vitro growth like
callus or suspension culture, embryo culture, anther culture etc. (Table – 1).

Table – 1: Types of Plant Tissue Culture

PLANT TISSUE
CULTURE DESCRIPTION
TYPE
Cell division in explants forms a callus. Callus is irregular, unorganized
and undifferentiated mass of actively dividing cells. Callus is obtained
Callus culture within 2-3 weeks in culture medium containing growth regulators like
auxins (e.g. 2,4-D) and cytokinins (e.g. BAP). Callus formation involves
three stages – induction, cell division and differentiation.
Suspension culture grows faster than callus culture. In this type of culture,
Suspension
single cells or group of cells are suspended in a liquid medium containing
culture
growth regulator like auxins (e.g. 2,4-D).
Shoot and root cultures are controlled by auxin-cytokinin ratio. High
Shoot and root
auxin promotes root culture whereas high cytokinin promotes shoot
culture
culture.
Embryo culture involves the process of extracting young immature
embryos from developing seed and growing them in vitro on culture
Embryo culture
medium to form seedlings. This is helpful in embryo rescue e.g. jute,
tomato, bean etc.
This involves culture of excised endosperm along with embryos from the
Endosperm immature seeds on suitable cultural medium. Triploid plants which
culture produce seedless fruits (e.g. apple, banana etc.) are developed from
endosperm culture.
Anther culture involves culture of pollens or anthers containing haploid
Anther culture microspores on suitable medium. It is important method in production of
haploid which are very useful in plant breeding.
Culturing of fertilized ovule on the usual basal medium. It is required in
Ovule culture those cases where embryo abort early and is difficult to excise from ovule
for embryo culture.
The ultimate objective in plant protoplast, cell and tissue culture is the reconstruction of plants
from the totipotent cell. On the other hand, the totipotentiallity of somatic cells has been
exploited in vegetative propagation of many economical, medicinal as well as agriculturally
important plant species.

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5.4. TOTIPOTENCY AND CONCEPT OF CELLULAR

DIFFERENTIATION
The basis of plant cell and tissue culture is ‘totipotency’. The term ‘totipotency’ is coined by T.
H. Morgan (1901) which indicates the capacity of cell to develop into an organism by
regeneration. The inherent potentiality of a plant cell to give rise to a whole plant is described as
cellular totipotency (L. totus = entire, potentia = power). In plants, mature and differentiated
cells can retain the ability to regenerate to a meristematic state as long as they are viable. In plant
tissue culture, we generally use explants (differentiated tissue) to initiate their growth in culture
or for callus culture. Since explant is a portion of mature tissue, so its non-dividing quiescent
cells undergo certain changes to regain its meristematic activity. This reversion of mature
differentiated cells to the meristematic state leading to the formation of undifferentiated callus
tissue is called ‘dedifferentiation’. The phenomenon of conversion of component cells of callus
to whole plant or plant organ is described as ‘redifferentiation’. These two phenomenon of
dedifferentiation and redifferentiation are described as ‘cellular totipotency’ which is found
only in plant cells and not in animal cells. The cells obtained from stem, root or other plant parts
are allowed to grow in culture medium containing mineral nutrients, vitamins and hormones
which produces an unorganized mass of cells which is known as callus tissue with totipotent
cells. Here, totipotency of the cell is manifested through the process of differentiation where
hormones play the major role than any other factors.
The basic event in the development of higher organisms is the specialization of cells, which is
termed as cytodifferentiation. In tissue culture, undifferentiated callus cells are all
parenchymatous in nature, so the differentiation of callus cells into a variety of cells is required
during redifferntiation of these cells. The efficient way for the study of cytodifferentiation in
vitro is the study of differentiation of callus parenchyma cells into vascular tissue e. g.
xylogenesis. Xylogenesis is the differentiation of parenchyma cells into cells with secondary
wall thickening as in xylem of vascular plants. It is shown that ‘sucrose’ and phytohormones like
auxins, cytokinins and gibberellins play important role in vascular differentiation. Several report
indicated that auxin is essential for cytodifferentiation and it effect vascular differentiation
quantitatively as well as qualitatively. Auxins at low concentration (0.5 mg per litre) is known to
stimulate xylem formation and its concentration show inverse relationship with the degree of
xylem differentiation. Auxins effect on vascular differentiation is also dependent on the presence
of sugar (sucrose) and with the variation in sugar concentration relative amount of xylem and
phloem formation can be altered. For eg., cell suspension culture of french bean when treated
with increased sugar concentration and cytokinin ratio to auxin, exhibits significant metabolic
changes (Robertson et al., 1995).
For the development of whole plant from callus totipotent cell, apart from cytodifferentiation,
there should be organogenesis i.e. formation of plant organs like stem, root, leaves and flower
etc. This can be accomplished either through ‘shoot bud differentiation’ or through ‘somatic
embryogenesis’. Former involves formation of shoot buds and latter involves somatic embryo
development and through both whole plant can be regenerated.

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Shoot bud differentiation is initiated by both chemical and physical factors and these factors
vary for different plant materials. In cultured tissue, besides genotypic influence shoot bud
differentiation is also dependent on the auxin/cytokinin ratio in the medium. The ratio of
cytokinin (kinetin) to auxin (indole acetic acid, IAA) plays major role in organogenesis of
tobacoo pith tissue. High kinetin level causes bud differentiation whereas high level of auxin
favours root differentiation. Some studies suggest that organ differentiation (bud or root
differentiation) was determined by the concentration of auxin (NAA) and not by the ratio of
cytokinin to auxin. Besides growth regulators, physical factors like light, temperature etc also
plays important role in organogenesis. High light intensity has been found to have inhibitory
effect on shoot bud differentiation in tobacco. Blue light promotes shoot bud differentiation
whereas red light stimulates rooting in tobacco callus (Bhojwani and Razdan, 1983). The size
and source of the explants also affects the shoot bud differentiation process. Larger the plant
material with parenchyma, cambium and vascular tissue, then the probability of shoot bud
formation is greater.
Somatic embryogenesis is an artificial process in which an embryo or a complete plant
development is initiated from a single somatic cell or a group of cells. It is an in vitro method of
plant regeneration which is used as biotechnological tool for sustained clonal propagation (Park
et al., 1998). In nature, embryo do not develop outside the ovules however instead of egg cell, it
may develop from nucellar cells or integuments of the ovule but it mature only in a special
environment of embryo sac and the same conditions should be maintained in culture for inducing
somatic embryogenesis. Somatic embryogenesis was studied in Daucus carota (carrots) by
Steward et al. (1958) where bipolar embryos developed from single cells. Later, this
phenomenon has been studied on other plants like Citrus (Kochba and Spiegel-Roy, 1977;
Gavish et al., 1991, 1992), Coffea sp. (Nakamura et al., 1992) and Zea mays (Emons and Kieft,
1991) etc. In vitro somatic embryogenesis is influenced by explant, growth regulators and
physical culture environment. Somatic embryogenesis either can be initiated directly from the
explants or indirectly by the establishment of callus (Suman and Kumar, 2016). Generally, for
somatic embryogenesis immature and less differentiated plant parts are used and in many cases
zygotic embryo in its early stage of development is used to initiate cultures as they possess
embryogenic competence and are termed as pre-embryogenic determined cells (PEDCs).
Embryogenesis is easy in suspension cultures, where presence of auxin in the medium is
generally essential for initiation of embryo. Mostly 2,4-D has been used to induce somatic
embryogenesis but in some cases, other auxins like NAA and IBA have also been used for this
purpose. On the other hand, phytohormones like cytokinins and gibberellins are known to have
inhibitory effect on potential embryonic cells. It has also been shown that a substantial amount of
nitrogen supplied in reduced form such as ammonium salts stimulate somatic embryogenesis
(Merkle et al., 1995). The source of nitrogen can be coconut milk, casein hydrolysate, amino
acids and ammonium ions (NH4Cl, NH4NO3).

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5.5. APPLICATION OF PLANT TISSUE CULTURE

Plant protoplast, cell and tissue cultures have wide application in several areas like agriculture,
horticulture forestry, floriculture, medicines and industries. It is an important tool for crop
improvement, micropropagation, production of disease resistant plant, somaclonal variation,
hybridization, genetic transformation, commercial production of natural compounds,
preservation of germplasm etc. (Fig. 1)

Fig. 1: Some of the application of plant tissue culture technology

Agriculture:
Now a day’s plant tissue culture is widely used in agriculture as it is the most efficient
technology in plant breeding for crop improvement. It has been used to create genetic variability
among crop plants to improve their health and to incorporate specific desirable traits through
gene transfer. Plant tissue culture is an important tool involved in micropropagation and this
technology has a wide application in crop improvement. The micropropagation technology has a
potential to producing superior quality plants, isolation of useful high yielding variants with
better disease resistance and stress tolerance capacities (Brown et al., 1995). Increase in yield up
to 150% of virus-free potatoes was obtained in controlled conditions (Singh et al., 1992). Clones
are produced through callus culture having inheritable characteristics different from those of
parent plants due to the occurrence of somaclonal variability (George et al., 1993). Haploid and
triploid production has an important use in plant breeding and improvement of crop plants.
Haploid is an easier system for induction of mutation and can be used for rapid selection of
mutants with desirable traits. Similarly, triploid plants raised through tissue culture are self
sterile and seedless. This characteristic increases the edibility of commercially important edible
fruits like apple, banana, grape, mulberry, watermelon etc.

Forestry:
Since time immemorial man had been utilizing forest resources to fulfil his basic requirements.
Today, rapid industrialization, urbanization and over-exploitation have led to depletion of forest

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resources. Now, it has become the major concern to increase the forest product to meet the need
of growing global population. Here, tissue culture techniques play the important role for the
production of quality planting stock. Micropropagation method is most extensively used in
forestry for rapid multiplication of important forest trees yielding high fuel, pulp, fruits or oil at a
large scale. Triploids of popular (Populus tremuloides) have better quality pulpwood. Therefore
it is important to the forest industry.

Medicine:
Today, with the advances in tissue culture techniques, molecular farming of various
pharmaceutical compounds like edible vaccines, antibiotics, diagnostic or therapeutic proteins,
secondary metabolites etc. has been started. For eg., there are some proteins that are used for
diagnosis of human disease (diagnostic proteins) whereas some proteins are used to cure diseases
(therapeutic proteins). By using tissue culture method, transgenic plant with foreign genes of
required proteins is produced in bioreactor on large scale at low cost. Similarly, many secondary
metabolites are harvested from plants for pharmaceutical medicines, for eg. anticancerous
compound taxol is obtained from Taxus sp. But these metabolites are naturally produced in plant
at low amount, here, micropropagation techniques are used for large scale production of these
plants in short period of time preventing over exploitation of plant species.

Industry:
In recent years plant tissue culture has become a major tool in production of compounds valuable
to the industry. Cultural practices are used in growth and maintenance of microorganisms
involved in industrial production of breweries, drugs, milk products etc. Higher plants are also
very important source of industrially important natural products like flavours, essential oils,
pigments, sweeteners etc. Genetic manipulation of cell cultures has great potential for altering
the metabolic profile of plants making them profitable to the industry. Transgenic plant cell
culture hold promise for industrial production of useful compounds like food additives, colours
etc. Plant cells can be cultured in fermenters for the industrial production of secondary
metabolites using cell culture. The success of plant cell or tissue culture has led to the many
novel possibilities and much advancement have been achieved in the area of micropropagation,
production of secondary metabolites, somatic hybridization, induction of haploid and production
of transgenic plants etc. Some of the above mentioned biotechnological techniques involving
tissue culture methods are discussed below.

5.6. CLONAL PROPAGATION OR MICROPROPAGATION

From the ancient time men have been vegetatively propagating variety of plant species for
fulfilling his need. Only during past few decades, this can be easily achieved through the
techniques of cell, tissues or organ culture and this process is known as clonal propagation or
micropropagation. Micropropagation is an alternative means of asexual propagation of
important plants through tissue culture or cell culture techniques. It offers many advantages over
traditional methods of plant propagation. Some of the major advantages are as follows:

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(i) Millions of clonal plants can be regenerated from small portion of tissue (explants).
(ii) Large number of plants can be produced in a short time and space.
(iii) Useful in developing disease free plants or developing resistance in many plant species.
(iv) Plants develop through this process are of same physiological age and hence they show
uniformity in growth and other traits.
(v) Continuous production of plants or its products round the year as it is independent of
seasonal changes.
(vi) Long time storage of important germplasm.
(vii) Provides disease free plant materials for international exchange.
Mostly micropropagation or clonal propagation is achieved by culturing sterilized shoot tips,
axillary buds, adventitious roots, somatic embryogenesis etc. This technique involves following
steps: Selection of explants and its surface sterilization, media selection and in vitro
establishment of tissue, shoots proliferation, rooting, conditioning of propagules, acclimatization
of plantlets in green house and finally transferring of plants to the fields (Fig. 2). Before
transferring to the field, plantlets should be transferred first to green house for acclimatization so
that they can easily survive sudden change in the environment and invasion by soil microbes in
natural field condition. This acclimatization requires many weeks and is followed by potting
plantlets into sterile peat or soil.

Fig. 2: Stages involved in Clonal propagation or Micropropagation

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Regeneration of plantlets through cell and tissue culture techniques has been achieved in wide
range of trees of high economic value. In recent years, many studies are aimed at large scale
micropropagation of important trees yielding fuel, pulp, timber, oils or fruits and through this
large number of valuable plant species have been successfully grown worldwide basis (Table –
2) (Gupta, 2019; Purohit, 2005).

Table – 2: List of some plant species grown in large scale through micropropagation
S. NO. CATEGORY PLANT SPECIES
Bambusa bambos, Dendrochalamus asper,
Dendroclamus strictus, Eucalyptus camaldulensis,
Agro-forestry plant
1 Eucalyptus citriodora, Eucalyptus terecticornis,
species
Leucaena spp., Populus deltoids, Populus
euphratica
Curcuma longa, Solanum tuberosum, zinziber
2 Cash crops
officinale
Alpinia, Bougainvillea, Callistemon,
Chrysanthemum Clerodendron inerme, Dahlia,
Dianthus, Dracaena, Duranta spp., Ficus
benjamine, Ficus spp., Gardenia, Geranium,
3 Ornamentals Hibiscus, Hydrangea, Iresine, Miscanthus,
Nandina, Peperomia, Petunia, Plumbago,
Philodendron scandens, Pogonantherum, Pothos,
Rosa spp., Thalictrum, Viola, Yucca.

Aloe vera, Artemisia spp., Bacopa monnieri,

Centella asiatica, Chlorophytum borivilianum,
Medicinal & Aromatic
4 Pelargonium spp., Pogostemon cablin, Swertia
plant species
chirata, Vanilla planifolia.

Allium spp., Asparagus spp., Carica papaya,

5 Fruit & vegetables Fragaria ananassa, Musa spp.

In India, the Department of Biotechnology (DBT), Government of India constituted a

“Conservation on Micropropagation Research and Technology Development (CMRTD)”,
which is involved in micropropagation related activities of various plant species, where it may
prove useful.
Advantages of Micropropagation:
1. Rapid multiplication of plants,
2. Large number of plantlets is obtained within a short period and from a small space.
3. Plants are obtained throughout the year, independent of seasons.
4. Since genetically similar plants or clones are produced by this method, therefore
desirable characters of superior variety are kept constant for many generations.
5. The rare and endangered species are saved by this method.

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5.7. ARTIFICIAL SEED

Through micropropagation, large number of propagules can be produced in limited time and
space but their storage and transportation for transplantation is a major problem. To solve this
problem, the concept of synthetic or artificial seeds has become popular. The concept of artificial
seed was first time given by T. Murashige of the U.S.A. at a Symposium in Belgium, in 1977.
Artificial or synthetic seeds are the somatic embryos encapsulated in a suitable matrix (e.g.
sodium alginate), along with substances like mycorrhizae, insecticides, fungicides and
herbicides. In these seeds, the protecting gel acts as seed coat and artificial endosperm providing
nutrient as in true seeds (Fig. 3). In India, this technique of synthetic seeds was standardized and
practiced for sandalwood and mulberry at BARC (Bombay).

Fig. 3: Diagram of Artificial seed or Synthetic seed

The production of encapsulated seeds involves following steps:

(a) Induce artificial embryos from cell suspension culture.
(b) Mix embryos well with 2% Na-alginate.
(c) Transfer embryos in a solution of calcium salt, Ca(NO3)2 for 30 minutes. Here,
individual embryo gets enclosed into clear and hardened beads of about 4 mm.
(d) Sieve the bead through a nylon mesh.
(e) Test the growth vigour of encapsulated embryos by plating in sand or soil amended
with pesticides.
Further, for increasing viability of embryo for longer time superfluro chemical oils are mixed
with gel which increases oxygen supply. There are four types of artificial seeds that have been
proposed – Coated desiccated somatic seeds (somatic embryos, coated with polyoxyethylene
and then desiccated, e.g. carrot, celery embryos etc.); Coated hydrated somatic seeds (coated
somatic embryos are hydrated by hydrating gels- sodium alginate, e.g. alfalfa, barley,
sandlewood etc.); Uncoated desiccated somatic seeds (uncoated somatic embryos are
desiccated, 8-15% moisture content, e.g. carrot, Glycine max etc.); Uncoated hydrated somatic
seeds (uncoated somatic embryos are hydrated through fluid drilling, e.g. carrot, tomato, lettuce

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etc.). S. L. Kitto and J. Janick (1985), produced citrus embryos in vitro. They tested 8
compounds for their synthetic coating properties on embryos and a polyethylene oxide (polyox
WSR-N75) revealed good encapsulating properties. Similarly, E. M. Muralidharan and A. F.
Mascarenhas in 1987 had done production of artificial seeds in Eucalyptus sp.

Advantages of Artificial seeds:

1. They can be stored for long period without loss of viability.
2. Easy to handle and transportation for transplantation.
3. They can be directly sown in the soil like natural seeds.
4. They do not need hardening in greenhouse.
High cost of production is the only limitation of artificial seeds but with further research and
advances surely this will go down and commercial production of synthetic seed will increase.

5.8. PRODUCTION OF HYBRIDS AND SOMACLONES

In 1960, E. C. Cocking at the University of Nottingham (U.K.) demonstrated that the protoplasts
can be obtained through enzymatic degradation of cell walls. This became a new tool of genetic
manipulation of plants and the fusion of protoplasts of genetically different species showing
physical or chemical incompatibility has also been possible.
Protoplast fusion or somatic hybridization is the most important use of protoplast culture and
is significant for hybridisation between species or genera which cannot be crossed through
conventional method of sexual hybridization. Somatic hybridization is playing an important role
in plant breeding and crop improvement. It has been possible to transfer useful genes (e.g. nif
genes, disease resistance genes) from one species to another, thus widening the genetic base for
plant breeding. Major aspects of protoplast fusion are - development of fertile amphidiploids
from sexually incompatible species; production of heterozygous lines within one plant species
e.g. potato; transfer of a part of genetic information from one species to another through
chromosomes elimination and the transfer of cytoplasmic genetic information from one to a
second species. During fusion of cytoplasm, the nuclei of two protoplasts may or may not fuse
together. When two nuclei present within a cell, and then this binucleate cell are known as
heterokaryon or heterocyte. When the two nuclei fuse during cytoplasmic fusion then the
resultant cell is known as hybrid or synkaryocyte and when the genetic information from one
of the two nuclei is lost during cytoplasmic fusion then the cells are known as cybrid or
cytoplasmic hybrid or heteroplast (Fig. 4) (Doods and Roberts, 1985; Dubey, 2016).
Production of cybrid is called cybridisation, while production of hybrid is called hybridisation.
Cytoplasmic genetic information controlled by cytoplasmic genes like male sterility in some
plants, susceptibility and resistance to some of the pathogens and drug etc. can be transferred
from one plant to another through cybrid formation and this information can be applicable in
plant breeding experiments. Cybrid technology has successfully been applied to carrot, Brassica
sp., Citrus, tobacco and sugar beet.

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Fig. 4: Hybrid/Cybrid production through protoplast fusion

Steps involved in somatic hybridisation are:
1. Isolation of protoplasts from suitable plants.
2. Mixing of protoplasts in centrifuge tube containing fugigenic chemicals like
polyethylene glycol (PEG), sodium nitrate (NaNO3), maintenance of high pH 10.5 and
temperature 37oC, resulting into viable heterokaryons.
3. Wall regeneration by heterokaryotic cells.
4. Fusion of nuclei to produce hybrid cells.
5. Plating and production of colonies of hybrid cells.
6. Selection of hybrid and induction of organogenesis.
7. Transfer of mature plants.
As mentioned above, after fusion of protoplasts wall regeneration takes place and cells undergo
mitosis. The resultant colonies are a mixed population of both homokaryotic fusion product and
hybrids. Hybrid cell should be differentiated from other cells through various selection methods
dependent on physical and biological properties of fused cells and their colonies. Somatic

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hybrids can be identified with the help of biochemical markers. P.S. Carlson and co-workers
(1972) produced first interspecific somatic hybrids between Nicotiana glauca and N. langsdorfii.
In 1978, Melchers and workers developed first intergeneric somatic hybrids between potato
(Solanum tuberosum) tomato (Lycopersicon esculantum) and which was known as pomato or
topato (Fig. 5) (Dubey, 2016).

Fig. 5: Hybrid plant (Pomato/Topato) produced through protoplast fusion of potato

and tomato plants
During plant tissue culture it is observed that genetic variability occurs spontaneously. P.J.
Larkin and W.R. Scowcroft (1981) coined a term ‘Somaclonal variation’ for indicating genetic
variability that occurs during plant tissue culture, and plant variants obtained is called
somaclones. According to them, this variation may be due to heterogeneity between cells and
explant tissue, spontaneous mutation and transposition of genetic materials in culture
environment. Evan et al. (1984) introduced the term gametoclonal variation for plant variant

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generated from gametic cells. Genetic variation occurs during in vitro culture and it may occur in
isolated protoplast, undifferentiated cells, calli, tissues etc. Variant selected from tissue cultures
have been referred as ‘calliclones’ (from callus cultures; Skirvin 1978) or ‘protoclones’ (from
protoplast culture; Shepard et al. 1980). The cause of variation is mostly due to chromosomal
alterations (change in structure or number of chromosomes) and there are several factors that
contribute to chromosomal alterations such as (i) pre-existing genetic variations in explants
tissues; (ii) occurrence of sponataneous mutations; (iii) Structural and numerical alterations in
chromosomes during in vitro culture; (iv) intracellular mutagenic agents produced during in vitro
growth; (v) activation of transposable elements (Bhaskaran, 1987; Larkin, P. J. and Scowcroft,
W. R., 1981; Razdan,2019) .
Somaclonal variation is one of the aspects of tissue culture and variation in characteristics of
somaclones indicates that it can be an additional tool for crop improvement. Possible advantage
of somaclonal variation over induced mutagenesis are – (i) frequency of variation is greater than
induced mutation; (ii) changes does not involve drastic genetic alterations; (iii) It occurs for trait
of both nuclear and cytoplasmic origin, so it has distinct advantage due to variations in
cytoplasmic genes. J. E. Shepard and co-workers (1980) screened about 100 somaclones of
Russet Burbank and found a significant and stable variation in its various traits - growth habit,
maturity, date, tuber uniformity, tuber skin colour and photoperiodic requirements and here
greater tuber uniformity and early onset of tuberization were agronomic improvements over the
parent variety. Thus, somaclonal variation has proved an alternative tool to plant breeding for
generating new varieties that can exhibit disease resistance and improvement in quality and yield
in many plants (Table-3) (Dubey, 2016; Purohit, 2005).

Table-3: Somaclonal variation in some crop plants

Crop Variant traits/Character
Brassica spp. Flowering time, precocious flowering from apex,
(Brassicaceae) stem or leaf, growth habit, gross morphology
Nicotiana tabacum Yield, plant height, leaf size, type of inflorescence,
(Solanaceae) alkaloids
Oryza sativa (Poaceae) Plant height, number of tillers per plant, panicle size,
seed fertility, flowering period.
Saccharum officinarum Cane diameter, stalk length and number, sugar yield,
(Poaceae) pathogenic disease
Solanum tuberosum Plant habit, disease resistance, shape, yield and
(Solanaceae) maturity period of tubers, photoperiod requirement,
tuber uniformity
Zea mays (Poaceae) Twin stalk formation, pollen fertility, endosperm and
seedling mutant

In India, Sugarcane Breeding Institute, Coimbatore has released many varieties produced
through the process of somaclonal variation, eg. sugarcane variety - Co 2001-13 (Sulabh) which
is resistant to red rot and drought. Bio-13 (somaclonal variant of Citronella java with 37% more
oil and 39% more citronellon) has been released for commercial cultivation by Central Institute

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for Medicinal and Aromatic Plants (CIMAP), Lucknow. Heinz Co. and DNA Plant Technology
Laboratories (USA) developed supertomatoes with high solid component which reduced
shipping and processing costs.

5.9. PRODUCTION OF SECONDARY METABOLITES/ NATURAL

PRODUCTS
The use of plant tissue culture for the production of useful compound has become a subject of
interest for biotechnologists in various biotechnological programmes. Microorganisms have
already been utilized in cultural practices for industrial production of drugs, breweries etc.
Similarly higher plants which are important source of natural products like pigments, sweeteners,
flavours, fragrances, antimicrobials etc. are also utilized in production of these compounds using
cultural techniques. Majority of these compounds belongs to a metabolic group called as
secondary metabolites and chemically these are of different types e.g. latex, alkaloids, tannins,
resins, terpentines etc. Secondary metabolites are generally not involved in vital metabolic
functions of plants but act as a chemical interface between plants and its surrounding
environment like protecting them from predators and attracting pollinators. These substances are
useful in combating many infectious disease or act as anticancer agents (Whitaker and
Hashimoto, 1986; Wink et al., 2005). As these compounds are naturally produced by plants in
small amount, therefore their production in large amount is done by cell suspension culture
technology. Plant cell culture has been considered as potential source of specific secondary
metabolites and cell cultures have following advantages in natural products/metabolite
production –
1. Rate of biochemical synthesis from small amount of plant material is quite high in
short time period.
2. Cultures are maintained in controlled conditions (environmental and nutritional)
ensuring continuous yields of metabolites.
3. A new means of product synthesis from plants difficult to grow or establish e.g.
quinine, pyrethroids etc.
4. A new route of noval compounds synthesis from deviant and mutant cell lines.
5. Some cell cultures have the capacity for biotransformation of specific substrate to
more valuable compounds. e.g. digoxin synthesis (Fowler, 1983).
6. Cell cultures are more economical for those plants which attain maturity very late
e.g. Papaver bracteatum (source of thebaine) attain maturity in 2-3 seasons.

Production of secondary metabolites can be achieved by selection of specific cells and the
development of a suitable medium. Through clonal selection such strains of cells are selected
that produces greater amount of these substances than those found in the intact plants. For this
purpose two methods are used – single cell cloning and cell aggregate cloning. The latter process
is easier than the first one as isolation and culture of single cell is comparatively difficult.
Nowadays in vitro production of useful compounds or secondary metabolites has increased and

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due to economical and pharmaceutical importance of these compounds, plant tissue culture
technology are utilized for large scale production of these substances (Table-4)(Dubey, 2016;
Razdan, 2019).

Table – 4: Secondary metabolites/natural products produced by plants and their uses -

S. Secondary metabolites/
Plant species Uses
no. Natural Products
1 Artemisia spp. Artemisin Antimalarial
Insecticidal, antiprotozoan,
2 Azadirachta indica Azadirachtin
anti-worm
Antibacterial, antiprotozoan,
3 Barbaris sp. Barberine
anti-inflamatory
4 Catharanthus roseus Vinblastine, vincristine Anticarcinogenic
5 Cinchona officinalis Quinine Antimalarial
6 Coffea arabica Caffeine Stimulant
7 Crocus sativa Saffron Flavouring/colouring agent
8 Digitalis lanata Digoxin Cardiac tonic
9 Dioscorea deltoidea Diosgenin Antifertility
10 Ephedra gerardiana Ephidrine Spasmolytic
11 Jasmium sp. Jasmine Perfume
12 Papaver sominiferum Codeine Sedative, analgesic
13 Rauwolfia serpentina Resperine Hypotensive
14 Taxus buccata Taxol Anticarcinogenic

.5.10 CRYOPRESERVATION & GERMPLASM PRESERVATION

The sum total of all genes present in a crop constitutes its germplasm or it refers to the heredity
material transmitted to offspring. In recent years, with the advances of modern tools and
technology, introduction of exotic species and due to the course of evolution many new
introduced plants species with desired traits have started replacing the primitive plants with some
valuable genetic traits. Hence, it is necessary to conserve these endangered species so that their
valuable traits can be retained. For this purpose, a global body namely International Board of
Plant Genetic Resources (IBPGR) has been established in 1974. Main objective of this
organization is to advance the conservation and use of plant genetic resources for the present and
future generations.
The main objective of germplasm conservation is to preserve the genetic diversity of plant for
its future use. There are two main approaches for germplasm conservation – in-situ and ex-situ
conservation. In-situ means conservation of germplasm in their natural environment i.e.
national parks, sanctuaries, biosphere reserves etc. Important aspect of this type of conservation

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is that it enables the natural evolutionary process among species allowing the appearance of new
recombinant forms. But this conservation process require controlled monitoring, thus have
expensive maintenance cost for large number of species. Also in nature, there is probability of
germplasm degradation due to various environmental hazards. Ex-situ means conservation of
genetic materials in form of seeds or in form of in vitro cultures (plant cells, tissues or organs
cultures) preserved in gene banks for long term storage under suitable conditions. In ex-situ
conservation, genetic resource is not conserved in its natural environment so it does not allow the
natural evolutionary process to continue, but it ensures the safety of genetic materials and
ensures its availability in need.
Traditionally, germplasm is conserved as seeds stored in required temperature. But conventional
methods fail to prevent losses due to disease, pathogens, pests, climatic disorders and other
economic causes. It could not save the viability of short lived seeds of economic plants like oil
palm, rubber, Citrus sp. and Coffea sp. (Dodds and Roberts, 1985). Biological activities of these
materials can be conserved for long time by storing at low temperature at which growth rate of
cells retards. It has been found very effective for tissue culture of many plant species such as
potato, cassava, pea, rice, wheat, coconut, palm, strawberry and sugarcane etc. There are three
main ways of in-vitro germplasm preservation: (i) Cryopreservation, (ii) Cold storage and (iii)
Low pressure and low oxygen storage.

I - CRYOPRESERVATION

Cryopreservation (gr. Kayos meaning “frost”) refers to preservation in the “frozen state”. It is the
process of storing cells, tissues or organs at very low temperature such as over solid carbon
dioxide (-79oC), in deep freezers (-80oC), in vapour phase nitrogen (-150oC) or in liquid nitrogen
(-196oC) in the presence of cryoprotectants. Various difficulties are also associated with
cryopreservation like certain features of plant cells (large size, water abundance and strong
vacuolization), cell damage during freezing and thawing process and large crystal formation that
often ruptures cell membranes etc. But due to presence of cryoprotectants these difficulties can
be overcome.

Steps of Cryopreservation:
(i) Selection of plant material – Preferential plant materials that are used in
cryopreservation are apical meristem, plant organs, protoplasts, anthers, pollen grains,
ovules and young embryos etc. Cultured cells are not ideal material for
cryopreservation.
(ii) Cryoprotectants – Addition of mixture of few cryoprotectors like dimethyl sulfoxide
(DMSO), sugars, sugar alcohols, alcohols, glycols, polyethylene glycol (PEG),
dextrins, glycerine, sucrose and some amino acids etc. These cryoprotectants protect
the preserved materials from cryodestruction.
(iii) Freezing of material – freezing of material should be done in regulated rate (desired
rate is commonly not less than 1oC per minute) in order to prevent intercellular
freezing and crystal formation. Types of freezing can be rapid, slow or stepwise.

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(iv) Storage of material in liquid N2 – For prolonged storage of frozen material very low
temperature is required and this can be achieved by the use of liquid N2 that maintains
the temperature at -196oC. At this temperature all the metabolic activities of cells get
retarded and it also prevents biochemical injury.
(v) Thawing and washing – This process involves the elevation of temperature (between
35-45oC) of frozen vials containing preserved material and as the last ice crystal
disappear, quickly transferring the vials to water bath (20-25oC). After thawing the
obtained plant materials should be washed to remove the toxic cryoprotectants. This
process involves – dilution, resuspension, centrifugation and removal of cells.
(vi) Reculturing – During above process there is possiblilty that some cells die due to
storage stress and strong one survive. In order to find out viable one there culturing on
growth medium is essential. Further, cell viability test can be done by using FDA
(Fluorescein Diacetate) staining and growth measurement (cell number, dry and fresh
weight, mitotic index).
Mitotic Index = Total number of dividing cells × 100
Total number of cells (dividing and undividing)
(vii) Regeneration – The selected viable cells are then cultured on growth media to
regenerate into plantlets.

II – COLD STORAGE
In this method, germplasm is conserve at a low and non-freezing temperature (1-9oC). In this
case growth activity of plant material is slowed down. This process prevents preserved materials
from cryogenic injuries and yields germplasm with good survival rate. It is simple and cost
effective process. Through this techniques grape plants and virus free strawberry have been
stored for fifteen years (9oC) and for six years (10oC).

III – LOW PRESSURE AND LOW OXYGEN STORAGE

In low pressure storage method, atmospheric pressure around the plant material is reduced
whereas in case of low oxygen storage method, the oxygen concentration is reduced which
ultimately reduces the in vitro growth of plants. This method is useful in increasing the shelf life
of many fruits, vegetables and flowers.

Significance of germplasm preservation

1. Preservation of genetic diversity.
2. Conserving plant materials of endangered species.
3. Long term conservation of cell cultures of several species through cryopreservation.
4. Preservation of disease free plant material that can be propagated when it is required.
5. Conservation of somaclonal variations.
6. Long term maintenance of recalcitrant seed.
7. Enhancing pollen longevity by conserving it.
8. Development of germplasm banks.

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9. Important in breeders programmes as it provide raw materials to breeder for producing

various crops.

The main limitation of germplasm storage is that this process requires expensive equipment and
trained persons for executing it in proper way. Organisations associated with germplasm are
International Plant Genetic Resources Institute (IPGRI), National Bureau of Plant Genetic
Resources (NBPGR), Forest Research Institute (FRI) and Botanical Survey of India (BSI).

5.11. SUMMARY
 Plant tissue culture is a collection of techniques used in maintaining and growing plant
cells, tissues or organs under sterile conditions on a nutrient medium.
 A German botanist, Gottlieb Haberlandt (1898,1902) has done intensive effort and
developed the concept of in vitro cell culture and is thus regarded as father of tissue
culture.
 In India, tissue culture work was pioneered by Panchanan Maheshwari who is regarded
as father of embryology in India.
 Guha and Maheshwari (1964-67), first time developed the haploid through anther and
pollen culture.
 Murashige and Skoog (1962) formulated MS medium suitable for many applications
and most widely used in plant tissue culture. Other media used in plant tissue culture are
proposed by Gamborg et al (1968), White (1963), and Smith (1967) etc.
 The general techniques used in plant tissue culture are cleaning of glassware, sterilization
of glassware and media, sterilization of plant material and culture.
 Totipotency is the basis of plant cell and tissue culture. The term ‘totipotency’ is coined
by T. H. Morgan (1901).
 The inherent potentiality of a plant cell to give rise to a whole plant is described as
cellular totipotency
 The reversion of mature differentiated cells of explant to the meristematic state -
undifferentiated callus tissue is called ‘dedifferentiation’. The conversion of component
cells of callus to whole plant or plant organ is described as ‘redifferentiation’. The
phenomenon of dedifferentiation and redifferentiation are described as ‘cellular
totipotency’ which is found only in plant cells.
 The basic event in the development of higher organisms is the specialization of cells i.e.
cytodifferentiation. For the development of whole plant from callus totipotent cell, there
should be organogenesis and this can be accomplished either through ‘shoot bud
differentiation’ or through ‘somatic embryogenesis’.
 Shoot bud differentiation involves formation of shoot buds and this is initiated by both
chemical and physical factors. Besides genotypic influence it is also dependent on the
auxin/cytokinin ratio in the medium.

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 Somatic embryogenesis is an in vitro method of plant regeneration in which an embryo

or a complete plant development is initiated from a single somatic cell or a group of cells
and this is widely used as important biotechnological tool.
 Somatic embryogenesis was reported in carrots by Steward et al. (1958) and later, It has
been studied on other plants like Citrus, Coffea sp. etc.
 Plant tissue cultures have wide application in several areas like agriculture, horticulture,
forestry etc. and it is an important tool for crop improvement, micropropagation,
production of disease resistant plant, commercial production of natural compounds,
conservation of germplasm etc.
 Micropropagation is an alternative means of asexual propagation of important plants
through tissue culture or cell culture techniques. It offers many advantages over
traditional methods of plant propagation like millions of clonal plants can be regenerated
from small explant in a short time and space, developing disease free plants, continuous
production of plants round the year etc.
 Artificial or synthetic seeds are the somatic embryos encapsulated in a suitable matrix
(e.g. sodium alginate), along with substances like mycorrhizae, insecticides, fungicides
and herbicides. The concept of artificial seed was first time given by T. Murashige
(1977).
 Protoplast fusion or somatic hybridization involves hybridisation between species or
genera during protoplast culture. It is playing an important role in plant breeding and crop
improvement.
 During protoplast fusion, when the two nuclei also fuse then the resultant cell is known as
hybrid or synkaryocyte and when the genetic information from one of the two nuclei is
lost then the cells are known as cybrid or cytoplasmic hybrid or heteroplast
 Somaclonal variation is the term used for indicating genetic variability that occurs
during plant tissue culture, and plant variants obtained is called somaclones. It is an
alternative tool to plant breeding for generating new plant varieties with useful traits like
disease resistance, improvement quality and high yield.
 Plant tissue culture is also used for the production of useful compound. Microorganisms
are utilized in industrial production of drugs, breweries etc. Similarly higher plants which
are source of natural products/ secondary metabolites are also utilized in production of
these compounds using cultural techniques.
 Germplasm refers to the sum total of all genes present in a crop. Through germplasm
conservation the genetic diversity of plant can be preserved for its future use.
 Germplasm conservation – in-situ and ex-situ conservation. In-situ - conservation of
germplasm in their natural environment (national parks, sanctuaries, biosphere reserves
etc). Ex-situ - conservation of genetic materials in form of seeds or in form of in vitro
cultures (plant cells, tissues or organs cultures) preserved in gene banks.
 There are three main ways of in-vitro germplasm preservation - Cryopreservation, cold
storage and low pressure and low oxygen storage.

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 Cryopreservation is the process of storing cells, tissues or organs at very low

temperature (in solid carbon dioxide.-79oC; deep freezers,-80oC; vapour phase nitrogen,-
150oC; liquid nitrogen,-196oC) in the presence of cryoprotectants.

5.12. GLOSSARY
Agar – a polysaccharide powder obtained from algae (e.g. Gracilaria, Gelidium) used to gel a
medium.

Anther culture – culture of single pollen grains or anther for producing monoploid plants.

Artificial seed – encapsulated somatic embryos that are treated like seed.

Aseptic – free of microorganisms; free of pathogens, contaminants, algae, bacteria, fungi,

viruses, etc.

Autoclave – a machine used for sterilizing wet or dry items with steam under pressure.

Auxin – Phytohormone that promotes callus growth, cell enlargement, adventitious buds and
lateral rooting.

Callus – mass of unorganized plant parenchyma cells.

Clone – asexually produced plant from a single source plant.

Clonal propagation – asexual production of genetically uniform plants from an explant.

Contamination – contaminated with unwanted microorganisms such as bacteria or fungi.

Cryopreservation – low temperature storage of cells, tissues, embryos, seeds etc.

Cryoprotectant – an agent that prevent freezing and thawing damage to cells during
cryopreservation.

Culture – in vitro growing of a plant.

Cybrid – a cytoplasmic hybrid cell with the nucleus of one fusing cell and cytoplasmic
organelles of another, or of both cells.

Cytokinin – a phytohormone that regulate growth, morphogenesis and cell division.

Differentiated – Cells with the specialized structure and function, typical of the cell type in
vivo.

Explant – the excised plant portion taken from its original site and transferred to culture medium
for growth or maintenance or for tissue culture.

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Gibberellins – a phytohormone that influences cell enlargement.

Hormones – growth regulators like cytokinins, auxins and gibberellins etc.

In vitro – to be grown within the glass or performed outside of living organism.

In vivo – to be grown naturally or within a living organism.

Medium – a nutritive solution for culturing cells.

Micropropagation – In vitro clonal propagation of plants from shoot tips, axillary buds,
adventitious roots etc.

Organ culture – culture of plant organs such as roots or shoots.

Somaclonal Variation – Phenotypic variation seen in plants (somaclones) produced by plant

tissue culture.

Somaclones – Plants obtained through cell culture by using somatic plant cells.

Somatic embryos – embryo like structure obtained from somatic cells.

Sterile – without life or a culture free from microorganisms.

Tissue culture – in vitro maintenance or growth of tissue (explant) in a culture medium under
sterile condition.

Totipotency – a potential of a cell to divide and give rise to entire plant.

Transgenic – plants having a piece of foreign DNA.

Undifferentiated – plant cells, existing in a state of cell development.

. 5.13. SELF ASSESSMENT QUESTION

I. Very Short Answer Type Questions:

Q1. Who is known as the father of plant tissue culture?

Q2. Who executed the experiments on wound healing in plants through callus formation?

Q3. Who prepared the first synthetic seeds?

Q4. Who produced first interspecific somatic hybrids between Nicotiana glauca and N.
langsdorfii?

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Q5. Conservation on Micropropagation Research and Technology Development (CMRTD) is

involved in ................ related activities of plant species.

Q6. Mention one of the main objectives of germplasm preservation?

Q7. Name any two varieties produced through the process of somaclonal variation?

Q8. What is the full form of NBPGR?

II. Multiple Choice Questions (MCQs):

Q1. What is the term used for unorganized mass of cells?

(a) Explants (c) Totipotency
(b) Callus (d) Regeneration
Q2. Who developed first haploids through anther and pollen culture?
(a) Guha and Maheshwari (1964) (c) T. H. Morgan (1901)
(b) Larkin and W.R. Scowcroft (1981) (d) None of Above
Q3. Which term is used for the ability of a cell to divide and regenerate into a whole plant?
(a) Callus (c) Regeneration
(b) Explants (d) Totipotency
.Q4. Haploid plants can be obtained from………culture.
(a) Bud culture (c) Root culture
(b) Leaf culture (d) Anther culture
Q5. What occurs at high auxin:cytokinin ratio?
(a) Somatic embryo (c) Root Initiation
(b) Callus (d) Shooting
Q6. What is the term used for the plant propagation through tissue culture?
(a) Hybrid (c) Micropropagation
(b) Cybrid (d) Regeneration
Q7. Name the chemicals used as fusogen during protoplast fusion.
(a) Polyethylene glycol (PEG) (c) MS Medium
(b) CaCl2 (d) None of Above
Q8. Who coined the term somaclonal variation?
(a) Larkin and W.R. Scowcroft (1981) (c) Guha and Maheshwari (1964)
(b) S. L. Kitto and J. Janick (1985) (d) Steward et al. (1958)
Q9. Which of the following substance is used as cryoprotectant?
(a) Nitrous oxide (c) Dimethyl sulfoxide (DMSO)
(b) MS Medium (d) None of Above
Q10. Most commonly used medium for culturing plant tissue.
(a) White medium (b) Gamborg medium

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(c) MS medium (d) Gautheret medium

Answers –
I. Very Short Answer Type Questions:
1. Gottlieb Haberlandt; 2. Henri-Louis Duhamel du Monceau; 3. Kitto and coworkers (1982); 4.
P.S. Carlson and co-workers (1972); 5. Micropropagation; 6. Save plants from extinction; 7.
Sugarcane variety - Co 2001-13 (Sulabh) and somaclonal variant of Citronella java - Bio-13; 8.
National Bureau of Plant Genetic Resources

II. Multiple Choice Questions (MCQs):

1. (b) Callus; 2. (a) Guha and Maheshwari (1964); 3. (d) Totipotency; 4. (d) Anther culture; 5.
(c) Root Initiation; 6. (c) Micropropagation; 7. (a) Polyethylene glycol (PEG); 8. (a) Larkin and
W.R. Scowcroft (1981); 9. (c) Dimethyl sulfoxide (DMSO); 10. (c) MS medium.

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embryogenesis. In: T.A. Thorpe (Ed.), In Vitro Embryogenesis in Plants. Kluwer, Dordrecht,
pp. 155-205.

Misawa, M. (1994). Plant tissue culture: An Alternative for production of Useful Metabolites.
FAO Agriculture Services Bull., Rome, 87 pp.

Morel, G. (1960). Producing virus-free Cymbidium. Am. Orchid Soc. Bull. 29: 495-497.

Muralidharan, E. M., Mascarenhas, A. F., (1987). In vitro plantlet formation by organogenesis in

E. camaldulensis and by somatic embryogenesis in E. citriodora. Plant Cell Reports 6, 256-
259.

Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco
tissue cultures, Physiologia Plantarum 15: 473-497.

Murashige, T., (1977). Plant Cell and Organ Cultures as Horticultural Practices. Symposium on
Tissue Culture for Horticultural Purposes, 78, 17-30.

Nakamura, T., Taniguchi, T., and Maeda, E., (1992). Studies on somatic embryogenesis in coffee
by scanning electron microscope. Jpn, J. Crop sci. 61: 476-486.

Park, Y.S.., Barrett, J. D. and Bonga, J. M. (1998). Application of somatic embryogenesis in high
value clonal forestry: development, genetic control and stability of cryopreserved clones. Cell.
Dev. Biol. Plant. 34: 231-239.

Purohit, S. S. (2005). Biotechnology Fundamentals and Applications, Agrobios (India), Jodhpur.

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Razdan, M. K. (2019). Introduction to plant tissue culture, Oxford & IBH Publishing Co. Pvt.
Ltd., New Delhi.

Robertson, D., Beech, I. and Bolwell, G. P., (1995). Regulation of the enzymes of UDP-sugar
metabolism during differentiation of French bean (Phaseolus vulgaris L.). Phytochemistry 39:
21-28.

Sato, F., Hashimoto, A., Hachiya, A., Tamara, K., Choi, K. B., Murashige, T., Fujimoto, H. and
Yamada, Y. (2001). Metabolic engineering of plant alkaloids biosynthesis. Proc, Natl. Acad.
Sci., USA 98: 367-372.

Shepard, J. F., Bidney, D. and Shahin, E. (1980). Potato protoplasts in crop improvement.
Science (N.Y.) 208: 17-24.

Singh, J. and Kumar, A. (2020). Plant Tissue Culture and Its Application in Agriculture as
Biotechnological Tool, Review Article, Int. J. Curr. Microbial. App. Sci., Special Issue-11:
274-284.

Skirvin, R. M. (1978). Natural and Induced variation in tissue culture, Euphytica 27: 241-266

Skoog, F. and Miller, C. O. (1957). Chemical regulation of growth and organ formation in plant
tissue cultured In Vitro. Symp. Soc. Exp. Biol. 11: 118-131.

Steward, F. C., Mapes, M. O., Mears, K., (1958). Growth and organised development of cultured
cells. II. Organizations in cultures grown from freely suspended cells. Am. J. Bot., 45: 705-
708.

Suman, S. and Kumar, H. (2016). Plant regeneration via somatic embryogenesis from the
cultured immature male floral buds of banana genotype ‘Dwarf Cavendish’. Natl. J. Life Sci.
13.

Whitaker, R. J. and Hashimoto, T., (1986). Production of secondary metabolites. In: D. A. Evans
et al. (eds.), Handbook of Plant Cell Culture, Vol. 4: Techniques and Applications. Macmillan
Publishing Co., N. Y., pp. 264-286.

Wink, A., Alfermann, A. W., Franke, R., Wetteraner, B., Distl, M., Windhovel, J., Krahn, O.,
Fuss, E., Garden, H., Mohagzadeh, A., Wildi, E. and Ripplinger,P., 2005. Sustainable
bioproduction of phytochemicals by plants in vitro cultures: anticancer agents. Plant Genetic
resour. Charact. Utill. 3: 90-100.

. 5.15. SUGGESTED READINGS

 A Textbook of Biotechnology – R. C. Dubey
 Elements of Biotechnology – P. K Gupta
 Introduction to plant tissue culture – M. K. Razdan

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 Biotechnology Fundamentals and Applications – S. S.Purohit

 Plant Tissue Culture: Theory and Practice - S. S. Bhojwani, and M. K. Razdan

. 5.16. TERMINAL QUESTIONS

Q1. Give a brief history of development of plant tissue culture.

Q2. What is micropropagation? What are its advantages over traditional methods of plant
propagation?

Q3. Give a brief account of ‘shoot bud differentiation’ and ‘somatic embryogenesis’

Q4. What is somatic hybridisation? Describe the various steps involved in somatic hybridisation.

Q5. What are cybrids and how these are produced? Mention some of the uses of cybrids in crop
improvement.

Q6. What is somaconal variation and how it can be induced? What are the advantages of
somaclonal variation over induced mutagenesis?

Q7. Give an account of production of secondary metabolites through plant cell culture.

Q8. What is cryopreservation? What are the steps involved in cryopreservation?

Q9. Discuss the application of plant tissue culture in agriculture, forestry, medicines and
industries.

Q10. Write a short note on the following?

(i) Callus
(ii) Totipotency
(iii) Cytodifferentiation
(iv) Steps of clonal propagation
(v) Artificial seeds
(vi) Protoplast fusion
(vii) Hybrid and Cybrid
(viii) Somaclonal varients
(ix) Germplasm conservation
(x) Cryoprotectants

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UNIT-6 ORGANOGENESIS AND ADVENTIVES

EMBRYOGENESIS

Contents:

6.1 Objectives
6.2 Introduction
6.3 Organogenesis and Adventives embryogenesis
6.4 Fundamental aspects of morphogenesis and androgenesis
6.5 Techniques
6.6 Utility
6.7 Summary
6.8 Glossary
6.9 Self Assessment Question
6.10 References
6.11 Suggested Readings
6.12 Terminal Questions

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6.1 OBJECTIVE

After reading this unit students will be able to-

• Understand the fundamentals of organogenesis and adventive embryogenesis.
• Understand the concept of morphogenesis.
• Understand the concept of androgenesis.
• Understand the technique of haploid production via anther culture and microspore
cultures.
• Understand the techniques and utility of these processes.

6.2 INTRODUCTION
The intact plant represents a highly organized and coordinate system in which number of
factors correlatively operates in integrated manner to form a plant with its several organs.
Regenerating complete plantlet from a single cell or tissue in vitro on an artificial culture
media is the one of the biggest achievement of the twentieth century. The technique which
deal with regeneration is called as “Plant tissue culture”, which is generally define as the
aseptic culture of tissue or cell in vitro. Plant tissue culture exploits the totipotent nature of
the plants and manipulates other conditions to regenerate the plantlets in vitro. Totipotency is
the ability of plant cell to perform all functions of development, which are characteristic of
zygote i.e its ability to develop into a complete plant. Besides this, this technique based on
two more fundamental process i.e., dedifferentiation and rededifferentiation, with the use of
some phytoregulators to regenerate plantlet. Primarily using plant tissue culture we
regenerate callus by using almost any part of the plant i.e., leaf, pith, root, floral part, petiole,
tubers, apical meristem etc and from every calli under appropriate conditions and
manipulations we may have organs of interest, embryo or complete plantlet.
This biotechnological approach or in other word bio- technological approach promise the
supply of plant throughout the year irrespect of season, further besides complete plantlet this
technique promise for producing organ (shoots or roots) of interest via organogenesis.
Organogenesis deals with an organ's initiation and development and we get organ of our
desire by just manipulating the culture condition. Besides the organ this technique also
provides a pure homozygous line in one generation or provides unlimited colons throughout
the time. Further, using this technique we may regenerate haploid, triploid, adventive
embryos which otherwise very tedious under natural conditions.
In this unit we are going to disuse some fundamental aspects, techniques and mechanism in
plant tissue culture.

6.3 ORGANOGENESIS AND ADVENTIVE EMBRYOGENESIS

6.3.1 Organogenesis
Plants, the eukaryotic multi-cellular organism consists of many specialized cell organizations.
Tissues and organs are among the many specialised cell groups seen in an adult plant.
Meristem, cortex, phloem, and epidermis are tissues made up of cells with a uniform shape

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and particular function. Leaves, roots, flowers, and the vascular system are examples of
tissues that are grouped together to form an organ.
Organogenesis is the process of an organ's initiation and development. Under normal in vivo
condition this process is under the control of number of genes (LEARY GENE, APETALA1,
APETALA2, APETALA3,PISTILLATA, AGAMOUS, STM, CLAVATA etc) and the expression
of each and every gene is under the control of provided conditions in the unit time
(temperature, humidity, season, water, stress etc).
During in vitro the induction of different adventitious organs of the plant from cultured
tissues under in vitro condition is known as organogenesis (Fig 6.1). The organogenesis
includes two steps, Caulogenesis (development of shoots) and rhizogenesis (development of
roots). Organogenesis begins with the production of primodium, which commences with the
development of a clump of meristematic cells termed meristamoids. Organogenesis begins in
the callus in response to the substances in the media stimulating it. Inducing organogenesis in
plant tissue culture is an important approach to regenerate plants from the culture. White
(1939) reported the first in vitro stimulation of shoot organogenesis using a tobacco hybrid,
while Nobecourt (1939) reported the first detection of root development using carrot callus.
The primary regulatory mechanism driving organogenesis was unknown until the late 1950s.
By growing explants, calli, and cell suspension in a specified medium, it is now able to attain
organogenesis in a significant variety of plant species. Organogenesis, which is basically
based on the concept of dedifferentiation and redifferentiation is a monopolar structure for
whole plant regeneration. It grows procambial threads that connect to the pre-existing
vascular tissue scattered throughout the callus or cultured explant. Organogenesis is
significantly more prevalent than somatic embryogenesis, and therefore has much more-
greater potential for clonal plant growth. Torrey (1966) proposed that organogenesis in callus
begins with the formation of a group of meristematic cells, that is, meristemoids, that can
respond to factors inside the system to generate a primordium, which can trigger the
formation of either shoot or embryoid depending on the types of inputs.

Organogenesis can be accomplished in two ways:

1. Direct organogenesis i.e., emergence of adventitious organs directly from explants.
2. In direct organogenesis i.e., development of organs passed via callus formation.

6.3.1.1- Organogenesis via callus formation

Callus is generally defined as the group of parenchymatous cells that are produced from the
explant via dedifferentiation and have potential for the redifferentiation. Organogenesis via
callus culture require, first establishment of successful aseptic culture from the selected
explants (cotyledons, hypocotyl, stem, leaf, shoot apex, root, immature inflorescence, flower
petals, petioles, embryos, etc) and then exposing the callus to different PGRs for
organogenesis. The explant may be in vitro grown or wild depending upon the conditions
and needs. Further, some time a specific explant may be needed for successful plant
propagation in any given species or variation. Callus formation can be induced in explants
from both mature and immature organs further; the explants with mitotically active cells are
usually preferred for callus induction. The explant's size and shape are also important aspects
during incubation. It has been discovered that just a tiny percentage of cells in a specific

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explant contribute to callus development. In explants peripheral surfaces or the excised

surface are the most common sites for callus formation. In vitro, callus is formed on explants
as a result of injury and in reaction to hormones provided in the medium, either endogenous
or exogenously. Because of their clonal qualities, culture survival, growth rates, and
totipotency in vitro, meristematic are always preferred over other tissues. Explants from
cereals such as meristems, shoot tips, axillary buds, immature leaves, and immature embryos
are particularly well suited. Explants from herbaceous species such as mature leaves, roots,
stems, petioles, and flower parts can often be effectively grown to generate plantlets through
organogenesis. The form of every callus is determined by the explant tissue or tissues from
which it emerged, as well as the medium employed to initiate and nourish it. Callus can be
sequentially subcultured and harvest for long periods of time, however the structure and
composition of the callus can alter over time as the medium favours certain cells and they
begin to dominate in the culture.
During organogenesis from the callus, the redifferentiation process came to take part and this
process is under the control of plant growth regulators and after any attempts and failures it
was established by Skoog and Miller (1957) and many other workers of the time that
successful redifferentiation can be achieved by just altering the concentrations of auxin and
cytokinin and according to their findings, a high ratio of auxin to cytokinin facilitated root
production whereas the opposite favoured shoot proliferation.

6.3.1.2- Direct Adventitious Organ formation

In some plants (generally woody) they have ability to give rise their clones directly during in
vitro cultures from their nodal explant and rarely from their apical shoot meristem. Besides
this, other ways of propagation is to induce adventitious shoots directly from intact bulb
scales, and basal disc (in case of globular herbs) particularly well suited to herbaceous
species in culture. Numerous examples of shoots developing accidentally on a variety of
organs have been documented in the literature across the plant world. Direct organogenesis
has been reported from stem segments in Acacia nilotica, hypocotyl explants has been
reported in Tamarindus indica. Direct organogeneis from cotyledon explants has been
reported in Tamarindus indica, Albizzia falcataria, and Sesbania grandiflora. Buds in
Begonias, for example, usually start along the injured leaf during in vitro culture.
The choice and nature of explant is further affecting the potential of direct organogenesis, in
some cases shoot production from explant (nodes or any other meristem) take place devoid of
growth regulator in the culture medium, but for other cytokinin are must be there in the
culture medium. Moreover, the amount of exogenous auxin and cytokinin required in the
process varies per tissue system, and appears to be dependent on endogenous hormone levels
in the tissue. These findings gave rise to the idea of totipotency, or the ability of all cells to
regenerate a whole new plant even after differentiation inside the plant's somatic tissues. As a
result, the reactivation of genes associated with the embryonic development stage will be
required for the production of adventitious structures. The rate of shoot formation from in
direct method in vitro regeneration may be significantly higher than that from the in direct
organogenesis, further these are the clone copy of the parent and rhizogenesis responses are
also high when placed in rooting cultures.

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Figure 6.1: Plant regeneration via Organogenesis

6.3.1.3- Mechanisms during organogenesis

Organogenesis is thought to occur by a process of differentiation in an undifferentiated mass
of parenchyma. The majority of parenchymatous cells are severely vacuolated, with small
nuclei and cytoplasm, and some have lignification. Random cell division would occur in this
set of cells, resulting in radial files of differentiated tissues. Meristematic centres, also known
as meristemoids, would arise as a result of these scattered cell division zones forming regions
of intense mitotic activity. These meristemoids might be found on the calli's surface or
embedded in the tissue. Continuing cell development in these meristemoids would result in
tiny protuberances on the calli's surface, giving the tissues a nodular look. According to
Torrey (1966), the primordia of organs develop either a shoot or a root from the meristemoids
by recurrent mitotic activity. Meristemoids are spherical masses of tiny isodiametric
meristematic cells with packed cytoplasm and a high nucleo-cytoplasmic ratio. Before
organogenesis, callus tissues store starch and other crystals, but these substances vanish
during meristemoid development. The cytoplasmic protrusions enter the vacuoles during the
early stages of meristemoid development, dispersing the vacuoles around the edge of each
cell or distributed all through the cytosol.

6.3.1.4- Factors affecting Organogenesis

The following factors directly or indirectly affects the process of organogenesis:

1. Explant Size- The size of the explant has a good impact on organogenesis. The
larger explant, which contains parenchymatous cell, vascular tissue and cambium, has
a higher regeneration capacity than the smaller explant.
2. Explant source- The explant source is one of the rate limiting and important aspects
of plant tissue culture, with the vast diversity of plants the source of organogenesis

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also vary from plant to plant. Depending on the species, the best part of the plant to
use for starting culture will vary. Like many plant species' leaves and leaf fragments
are good for callus induction but in some other petiole is best. Such as Begonia,
Solanum, Nicotiana, Crepis, and others have the ability to regenerate the shoot buds
from leaves explants. In Thymus Spp nodal part is of preference for shoot
regeneration. Likewise, bud production capacity is high in many monocot species
with specialised storage organs.
3. Explant Age- The age of the explant is another important factor that affects
organogenesis. In the case of Nicotiana sp., regeneration of an adventitious shoot bud
is only observed when leaf explants are gathered during the vegetative phase, prior to
flowering. Young Echeveria sp. leaf explants produce just roots, whereas older leaf
explants produce only shoot buds, and medium-aged leaves produce both shoots and
roots.
4. Light quality and intensity-Shoot formation is promoted by blue light, while
rooting is induced by red light. The organogenesis phenomena, is also stimulated by
the application of blue light followed by red light. Artificial fluorescence light
promotes rooting in some cultures while inhibiting it in others. In the case of Pisum
sativum, shoot bud initiation occurs in the dark, followed by a brief exposure to light.
5. Temperature-The majority of tissue cultures can be successfully cultivated at twp.
around 25 oC. The ideal temperature for a variety of bulbous species could be as low
as 15-18°C. Increases in temperature up to 33oC have been linked to increased
tobacco callus formation, however for shoot bud initiation, a lower temperature of
around 18 oC may be best.
6. Culture Medium-Although some reports show the production of leaf shoot buds on
culture cultivated in a liquid medium, medium solidified with agar favours bud
formation.
7. Medium pH- Organogenesis may be influenced by pH. Before sterilisation, the PH
of the culture medium is usually adjusted to 5.6 to 5.8.
8. Ploidy level- Chromosomal stability is the most crucial component in sustaining
callus tissue's organogenic potential. Ploidy level (Anuploidy, polyploidy, and other
chromosome number variations) in cultured plant cells have been thoroughly
described and it is observed that there is a general decrease in the morphogenetic
potentiality of callus tissue as chromosomal instability increases. The morphogenetic
capacity of a totipotent callus may be reduced or even lost during repeated culturing.
Other cultivated tissues with a genetic imbalance have been documented, including
pollen from Ginkgo, and stalk callus in Tobacco.
9. Age of Culture- Organs are typically produced by a young culture. However, in old
culture, the organogenic potential may decline and eventually vanish. Plant
regeneration potential can be maintained endlessly for many years in specific cultures
of some plants.
10. Chemical factors (Phytohormones)-

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a. Ratio of Auxin to Cytokinin: A high ratio of auxin to cytokinin promotes root

development, whereas an elevated concentration of cytokinin and low auxin
promotes shoot development.
b. Auxin in low concentration: The removal of auxin after pre-culture results in
root development. Auxin 2.4-D is a powerful growth agent that is metabolised
slower by tissues and hence stays in the medium longer than IAA or NAA. It is
also more active in activating meristematic activity, which leads to cell
proliferation. Whatever the type of the culture system utilised, tissue growth on a
2,4-D medium for a period of time followed by transfer to a 2,4-D free media
causes differentiation/organogenesis. In most cases, the loss of morphogenetic
potential is linked to 'habituation,' a condition associated with cytokinin-
autotrophy in long-term cultures.

6.3.2- Adventive embryogenesis

Embryogenesis, a general term use to explain the one of the basic fundamental process of
reproductive biology, which include’s the syngamy followed by development of the embryo
from the zygote. But some time embryos may developed from other ovular tissue i.e.,
nucellus and integuments or rarely from cells of embryo sac, since these cells are haploid in
nature the embryo produced is diploid and called as somatic embryo, adventive embryo,
embyoid, adventitious embryo and supernumerary embryo. These type of embryos naturally
reported in Citrus, Mangifera, Optunia, Limnathes etc, and the path and factor contributing in
the production is studied experimentally in Citrus by many workers.
As an alternative, the plant tissue culture technique exploiting the totipotent nature of plant
cells to regenerate the plants and in this way it was reported that not only ovular tissue but all
other somatic cells may have the potential to produce the embryos and the in vitro produced
embryos are called as somatic embryos. The initiation and development of embryos from
somatic tissues in plant tissue culture was first recognised by Steward et.al., 1958 and
Reinert, 1958-1959 in Daucus carota.

Majorly the somatic embryos are in demand due to the following reason
1. Large Scale Propagation Compared to Zygotic Embryos
2. Feasibility of single cell origin and the possibility of automating the large-scale
production of embryos in bioreactors.
3. Production of synthetic seeds from these somatic embryos.
4. This technique is more useful than Organogenesis and use in mutant production.

6.4 FUNDAMENTAL ASPECTS OF MORPHOGENESIS AND

ANDROGENESIS

6.4.1- Morphogenesis
Plant morphogenesis is a biological mechanism in which plant assumes its specific forms in
relation to time (pre to post) during development under the expression of both external and
internal signalling. In other word this process in under the controls of the spatial patterns of

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cells during embryo development to cause a tissue or organ to establish its shape.
Morphogenesis command tissue organisation, which affects a lot of an organisms anatomy,
physiology, and behaviour. This process involves in integrating growth and differentiation
through cell division and specialisation as a consequence of spatial and temporal hormonal
control mediated by the regulation and expression of multiple gene systems, the relating
action of meristem and their derivatives, and environmental variations. Plant hormones and
mineral nutrient requirements for morphogenetic responses were considered to be the only
important aspect of the morphogenesis. But later it was realized that not only hormonal and
nutritional interaction, but physiological interaction at cellular levels are also essential to
understand morphogenesis.

Plants pass through three phases of development (morphogenic competence, development

determination, and morphological differentiation) at both at the cellular level and in tissues.
The morphogenic competence is defined as the cell's ability to recognize a specific signal that
leads to a particular development under control conditions. Further, competent cells are
decided by induction forcing them to reroute their development as per the received signals,
which results, some of the cells differentiate, resulting in a new tissue organisation. Extrinsic
variables, whether biotic or abiotic, regulate the process of morphogenesis in addition to a
number of cell intrinsic factors. These factors will function by either influencing cell activity
to direct a specific development in a defined way, or by converting cells to restore
their totipotent traits and then again using their totipotenty nature to remodelling if required.
Apart from the plant growth regulator, the morphogenic process includes cell competence,
polarity, habituation, and gene control performance. So, Morphogenesis can be induced by
the combination of chemical as well as mechanical factors.

6.4.1.2- Cell Competence

The morphogenesis process, such as the growth of new cell structures, is closely linked to the
cell's ability to respond to extrinsic and intrinsic signals, which begins with the rupturing of
cell determination and the first cell divisions that give rise to meristemoids. Meristems'
ability to generate a new organism from an explant is determined by several steps, including
competence acquisition, induction or morphogenic commitment to a certain route,
differentiation, and eventually development. There is a direct link between a cell's ability to
generate different cell types and its degree of dedifferentiation and morphogenic competence.
So, the cells can be classified as multipotent, totipotent, or pluripotent depending on the
degree of dedifferentiation.

6.4.1.3- Cellular determination

Cellular determination is the process by which a cell's development competence is limited to
a specific route that is dependent prior to acquisition or a target cell's ability to respond
toward the specific developmental signals, such as metabolic, molecular, or hormonal
signalling and cellular positioning. The formation of cell polarity, asymmetric division, and
cell placement in the plant body are all required for differentiation. In tissue culture,
however, the extent of differentiation can be changed, causing the cells to become less
differentiated. This happens when cells are released from the body's regulation and exposed

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to a new situation in the growth media, causing them to dedifferentiate and display their
genome in a different way, resulting in novel patterns of differentiation and the formation of
new ordered structures. The age of the cell and its degree of differentiation, determination,
and/or residual memory retention of the original somatic cell have a direct influence on the
dedifferentiation and subsequent differentiation and development of new cellular lineages
Cellular differentiation is defined by Moore (1979) as the change of genetically similar cells
generated from a zygote or any other cell into biochemically, physiologically, and
architecturally differentiated cells. Finally, cellular differentiation is a result of at least three
factors: genetics, which is established at fertilisation, and incorporation of "stock" of
potential, which may be expressed throughout progression; ontogeny, which began as a
response to environmental stimuli but, once formed, tends to remain in a consistent condition;
and attributes, which originated in ontogeny, initially as a response to environmental stimuli
but, once established, tend to remain in a stable state.

6.4.1.4- Cellular habituation

Habituation is a persistent and hereditary decrease of growth factor needed by grown plant
cells. According to Meins (1989), auxin and cytokinin cellular habituation is caused by
transitory changes in cellular heredity known as epigenetic alterations. Epigenetic
modifications, in comparison to mutations, are reversible and directed, that is, they occur in
response to a specific inductor and suggest alterations in DNA that influence gene expression.
The stage of growth of the cells has a substantial influence on the inclination to habituation
and the competence for cytokinin habituation varies by tissue.
The main obstacle for commercial production may be cellular habituation associated with
extended periods of in vitro subcultures, as a result of the progressive loss of plant vigour for
example, tobacco callus habituated to cytokinin, where it has been shown that the higher the
amount of these endogenous substances, the lesser the capacity to develop adventitious buds,
implying an inversely proportional relationship for the both procedures. Because it endures
when cells are cloned and is relatively durable, the habituated state entails a change in
cellular heredity. When habituated cells may be forced to form full they may revert to the
cytokinin-requiring condition, indicating expression of a pre-existing potentiality. These
tissues are frequently identical to fully changed crown gall tissues and hereditary tumour
tissues, and they are capable of manufacturing large amounts of the growth factor to which
they have become accustomed. This disorder is thought to be caused by somatic mutations in
the gene complex that controls hormone regulatory systems, as well as chromosomal
anomalies.

6.4.1.5- Growth regulators in Morphogenesis

Plant growth regulators (endogenous or either applied or exogenous) have a direct influence
in morphogenesis of plants, which is different for different species and in different ways.
Auxin one of the dynamic plant hormone a variety of developmental processes and adjust the
growth and morphology of plants according to external environmental conditions, few
examples for auxins are apical dominance, the induction of vascular differentiation and the
retardation of leaf abscission and fruit development are also controlled by the levels of auxin
and many other process transportation of organic solute which indirectly contribute in

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morphogenesis. Number of similar effects has been reported for cytokinins also. In
conclusion, these growth regulators have their role in morphogenesis as they are involve in
altering the gene expression.

Further, exposure the culture to various growth regulator or their combinations triggers a pre-
programmed series of events within the cell, culminating in a heavily regulated sequence of
cellular division that leads to the creation of root or shoot primordia. Partially structured
meristematic aggregates can occur, which then get obstructed at a later stage of development
and growth. To bypass this stumbling block, the meristemoids may need to be withdrawn
from the original medium and transplanted to a new medium that contains either a different
combination of growth regulators or none at all. Even if a growth substance's morphogenetic
effects in cultured cells are well known, it is currently hard to provide adequate explanations
for the mechanism of its action in formation.

Many, though not all, of the following features must be included in any understanding of the
role of growth regulators in morphogenesis.
a. In most cases, growth regulators have a generic impact. Even distinct growth
regulators from the same family can cause morphogenetic changes in the same tissue.
b. The amalgamation or more growth regulators at sufficient levels or in certain ratios
causes some morphogenetic responses, whereas other doses of the same growth
regulators have no impact or have a markedly distinct effect.
c. In most plant cell cultures, there is a lag period of several days to several weeks
between the application of growth regulators and the morphogenetic action.
d. In some circumstances, sequential dosing with multiple growth factors is more
successful than simultaneous treatment with the same growth regulators in inducing
morphogenesis.
e. The response of cells from the same plant or cells growing in vitro to the same
growth regulators varies significantly over time.

Growth regulators are thought to regulate morphogenesis through the following mechanisms:
a. Inside the cell, by using relative or absolute concentrators of two or more growth
regulators.
b. Through the growth regulator's metabolism.
c. Through interaction with particular receptors at the membrane level.
d. Through indirect effects on nutrient uptake
e. But majorly at gene expression level.

6.4.1.6- Specific receptor interactions

The production of new shoot and root primordia from callus cells does not always necessitate
the formation of many different kinds with radically different sets of proteins, nor does it
necessitate a significant shift in cell division rate. The disorganised callus mass was separated
from the root or shoot primodium by the orderly pattern of cell wall development.
Membranes have been proposed as a primary target for growth regulator activity in a number
of ways, the majority of which are dependent on the presence of certain receptors. Even

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though the binding of numerous growth regulators to presumed proteinaceous receptors on a

surface has been proven in several plant tissues, it is currently unknown how such
associations induce the change in cell division pattern that eventually leads to the creation of
an ordered meristem. The metabolic machinery of the cell must be ready to understand the
signals resulting from the interaction of the two in order to activate these reactions. The
growth regulator is usually thought to be a limiting factor when an exogenously provided
growth regulator generates a morphogenetic response, however this isn't always the case.
Although the mechanism governing the number and distribution of receptors on the
membrane is unknown, a variety of unrelated factors such as minerals, temperature, osmotic
stress, light, sugars, and genetic and intrinsic factors that affect specific growth and
morphogenetic processes may all play a role. Treatment with growth regulators may
potentially affect the number of receptor sites on the cell, similar to what is seen in animal
cell systems.

Modifications of the affinity of the receptors for the growth regulator is another mechanism
by which cell sensitivity can be modified in response to varied external variables, such as
varying concentrations of the same growth regulator. It's possible that the receptor is a
complex protein that can go through one or more structural changes based on the growth
regulator's concentration in the environment. Moreover, the varying affinity of the receptor
for these compounds could explain differences in sensitivity of cells to different growth
regulators of the same class.

6.4.2 Androgenesis
Sporophyte is the resultant of fertilization of male and female gamete and contains a set of
chromosomes from each parent with genomic constitution of 2n (Figure 6.2). Haploids plants
are defined as sporophytes having only a single set of chromosomes. The ability of plant
regeneration directly from anther or microspore culture under in vitro conditions is referred to
as androgenesis which is of tremendous importance in plant breeding, plant physiology and
embryology studies because either spontaneous doubling or an application of the chemical
colchicines to double the chromosome number gives rise to homozygous plant in a single
generation. Further, androgenesis is based on the notion of stopping pollen cells from
developing into sexual cells and forcing them to develop directly into a mature plant via
embryogenesis or developed into calli and then into a plant. Till date it’s reported in many
families majorly dominating families Solanaceae and Poaceae. Besides, the androgenic
methods, gynogenic method are also in practice to produce homozygous plant but gynogenic
method gave result inferior than the androgenesis. Immature pollens are driven to pursue the
sporophytic mode of development in Androgenesis by a variety of physical and chemical
stimuli. The essential premise is to prevent pollen cells from developing into gametes, or
sexual cells, and instead drive them to develop directly into plants. Extracted anthers and
separated pollen can both be cultured to produce haploids.

6.4.2.1- Mechanism of Anther Culture-

It is the process of using anthers in the culture to produce haploid plantlets. The technique
was discovered by Guha and Maheshwari in 1964. Anther culture success is determined by

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the variety chosen, the plant's growing condition, and the quality of the donor material.
Natural flowering settings are usually the ideal for donor plants to yield anthers that can be
used in regeneration trials.

1. Pre-treatment of anthers- The technique of anther culture is rather simple, quick

and efficient. The selected microspore requires specific pre-treatment conditions for
anther callus induction. Young flower buds with immature anthers are surface
sterilised and rinsed with sterile water to ensure that the microspores are confined
within the anther sac at the right stage of pollen formation. Flamed forceps are used to
extract the calyx from the flower bud. The stamens are removed and placed in a sterile
petri dish after the corolla is sliced open. To determine the stage of pollen
development, one of the anthers is crushed in acetocarmine. Each anther is gently
detached from the filament and the intact undamaged anthers are injected horizontally
on nutrient media if it is confirmed to be in the correct stage. When working with
plants with small blooms, such as Brassica and Trifolium, a stereo microscope may be
required to dissect the anthers.
2. Media and Growth regulators- The medium requirement may vary with the type
of specie used. The most commonly used medium for anther culture is MS
(Murashige and Skoog, 1962) medium. The constituents and basal media, as well as
the combinations of growth regulators, all play a role in effective androgenesis. Agar
is used to solidify anther culture media. In some species, agar may include chemicals
that hinder the androgenic process. Some studies have proposed for the use of liquid
medium as a strategy to circumvent possibly inhibiting components in gelly agents.
The wall tissue of responding anthers turns dark over time, and within 3-8 weeks, they
break open due to pressure applied by the expanding pollen callus or pollen plants.
Individual plantlets or shoots coming from the callus are detached and put to a
medium that will allow further development after they reach a height of about 3-5 cm.
In the pots, the rooted plants are transferred to sterile soil mix. Most species required
a comprehensive nutritional medium (mineral salt, vitamins, sucrose) as well as
growth regulators during androgenesis. Auxin, on the other hand, is only required in
low concentrations in the medium in most species. Auxin, which is required for pollen
development and callus formation, is occasionally employed in conjunction with
Cytokinin. Other ingredients such as glutamine, cesine, proline, biotin, ionositol,
coconut water, silver nitrate, and polyvinylpyrrolidon can be added. Adding
glutamine and glutathione to the culture medium improves the embryogenic process
as well.
3. Induction and stages of Pollen development: After inoculation, haploid plants
develop from anther culture either directly or indirectly (through callus phase).
3.1 Direct Androgenesis- It is also known as pollen derived androgenesis. In this
case, pollen grains act as zygotes and go through numerous embryogenic stages
that are analogous to embryogenesis.
3.2 Indirect Androgenesis- Instead of typical androgenesis, the pollen grain
divides to form callus and then the callus further differentiate into shoots and roots

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in appropriate medium. This type of androgenesis is reported in Rice, tomato, and

wheat where callus tissue redifferentiated to generate haploid plantlets.

6.4.2.2- Mechanism of Microspore Culture:

During the microspore culture, anthers are gathered from sterilised flower buds in a tiny
beaker with basal medium in a standard process for microspore culture (e.g 50 anthers of
Nicotiana in 10 ml media, fig 6.3). The microspores are then pushed out of the anthers using
a glass rod against the side of the beaker. Filter the suspension through a nylon sieve, with a
pore diameter slightly larger than the diameter of the pollen and then removes residue from
the suspension. It has been observed that smaller microspores do not regenerate, thus larger,
good and viable microspores can be concentrated by filtering the microspore suspension
through nylon sieves. This pollen solution is then agitated for 5 minutes at a low speed of
150× g. The fine debris-containing supernatant is removed, and the pollen pellet is
resuspended in fresh medium and washed at least twice. After that, the microspores are mixed
with a suitable culture medium at a density of 103-104 microspores/ml. Pipette the completed
suspension into miniature petridishes. The liquid layer in the disc must be as thin to promote
proper aeration. To avoid dehydration, each dish is then wrapped with parafilm and
incubated. The responding microspores form embryos, or calli, which can then be transferred
to suitable environments for further growth into plants.

Microspore culture have some advantage over the anther culture, because during anther
culture this has been reported that some time if proper screening is not conducted or some
other reason the callus from the other sections of the anthers get involved in the cultures then
the resulting population of plants show various ploidy levels. This problem can be solved by
cultivating isolated microspores, which has the following benefits:
1. The anther wall and other accompanying tissues have no uncontrollable
consequences, and many parameters influencing androgenesis can be better regulated.
However, where the anther wall has a stimulatory impact, this is a disadvantage.
2. Starting with a single cell, the sequence of androgenesis can be observed.
3. Because microspores can be equally exposed to chemicals or physical mutagens, they
are appropriate for absorption, transformation, and mutagenic research.
4. Higher plant yields per anther could be achieved.

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Figure 6.2: Androgenesis via anther culture

Figure 6.3: Plant regeneration via androgenesis through microspore culture

6.4.2.3- Factors affecting Androgenesis:

1. Physiological Status of the donor plant- The age and conditions in which the plant
was grown have an impact on its physiology, and this feature of the donor plant has an
impact on in vitro androgenesis. Flowers that bloom early, for example, have a stronger
response to culture than flowers that bloom later. In anthers excised near the end of the
season, sporophyte development has also been seen in a low frequency. In some plants
(such as Brassica rapa), anthers removed at the old and sticky phases produce more
embryos than anthers removed at the juvenile stage. A stress situation or treatment of
plants with substances such as feminising agents and gametocidal compounds (which
interfere with normal growth) in some other plants.The differences in anther responses
from plants growing under various environmental circumstances could be attributed to
differences in endogenous growth regulator levels. Light intensity, temperature,

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photoperiod, nutrition, and carbon dioxide content are all important environmental
elements.
2. Stage of Pollen-It has been determined that the stage of anther selection is the most
crucial. Microspores ranging from tetrad to binucleate are sensitive in anthers. However,
once starch deposition begins in the microspore, there is no sporophytic development
and, as a result, no macroscopic structure construction.
3. Anther walls-The epidermis, endothecium, middle layers, and tapetum make up the
anther wall of pollens. The efficiency of the anther wall in pollen cultures has been
demonstrated in several investigations. Even when pollen from one cultivar of a plant is
introduced to the anther cultures of another cultivar, it develops into an embryo. Other
investigations have found that anther extracts in pollen cultures improve productivity and
embryo development. The nursing nature of anther walls in pollen cultures has been
proven to be an important element in androgenic response in these investigations.
4. Genotype-Plant genotype is one of the most important elements influencing
proliferation and differentiation in microscopic cultures. Some plant species respond
more positively to pollen-derived embryogenesis than others. Japanese rice, for example,
has a higher androgenic reaction than indica rice. Some plant hybrids exhibit a wide
range of responses. Some hybrids have a stronger androgenic response than others,
whereas others are unresponsive. The interaction between the genotype of the plant and
the environment in which this is raised could explain the heterogeneity of microscopic
culture responses. Some plants' poor-responsive lines (such as Solanum tuberosum) may
be intercrossed to develop lines with better androgenic responses. In tetraploid
Melandrium, however, the presence of X-chromosomes enhances the androgenic
response, whereas the presence of even one chromosome decreases it.
5. Pre-treatment of Anthers-The androgenic response is enhanced when anther/pollen
culture is pre-treated.
The embryo yield is increased when Nicotiana tabacum anthers/pollen are cold treated (at
5°C for 72 hours) before being heated to 25 °C. White burley buds treated at 7-9 °C are
more effective than those treated at 5 °C. After a high-temperature shock of 30-35°C for
1-4 days, some Brassica species and wheat genotypes show a higher androgenic
response.Cold treatment followed by centrifugation improves androgenic anthers as well
as the quick and synchronised development of embryos in some species.
Low-dose irradiation of anthers before culture has been reported to stimulate callusing and
embryogenesis in several plants, such as Datura and Nicotiana species.
6. Culture Media-In tobacco, adding ethereal to the nutrient culture mix or putting
excised anthers on agar-sucrose plates has been shown to increase the androgenic
response. The inclusion of sucrose in various quantities has been shown to aid pollen
callus formation. Potato and wheat, for example, require 6% sucrose, while Brassica
plants require 12-13 percent. For optimal embryo growth, iron is essential in the culture
media during the post-inductive stages of anthers culture. Low nitrogen levels in the
media have been proven to enhance androgenesis in some studies.

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7. Light-Light is not required for the growth of anther cultures. Incubation of cultures in
the dark for the first 24 hours followed by diffuse lighting was found to induce
embryogenesis in anther cultures.
8. Culture Density-Many studies have indicated that a minimum culture density of 3000
pollen/ml of growth media is needed for embryogenesis, but that a culture densities of
10000-40000 pollen/ml of culture medium produces the maximum embryo yield.

6.5 Techniques

6.5.1- General technique in in-vitro cultures.

Stage-0 (Pre-propagation stage)
The pre-propagation stage requires proper maintenance of the mother plants in the
greenhouse under disease and insect free conditions with minimal dust. Clean enclosed areas,
glasshouses, plastic tunnels and net covered tunnels, provide high quality explant source
plants with minimal infection. Collection of explants for clonal propagation should be done
after appropriate pre-treatment of the mother plants with fungicides and pesticides to
minimize contamination in the in vitro cultures. This improves the growth and multiplication
rates of in vitro cultures. The control of contamination begins with the pre-treatment of the
donor plants. The choice of explant depends on the methods.

Stage-I (Initiation of aseptic culture)

In this stage sterilization of explants and establishment of explants were done. The plant
organ used to initiate a culture is called explant. For sterilization number of different methods
and chemicals are available and they are used depending upon the need and utility. Further,
the choice of explant depends on the methods to be followed.
1. For organogenesis it may be leaf, node, root, or any part.
2. For androgenesis anther or pollen is choice.
3. For somatic embryogenesis callus from leaves, petiole or any other tissue or some
time it might be from single cell.

Stage-II (Multiplication of culture)

The success of any in vitro technique is largely dependent on the efficiency of this stage. In
this stage multiplication of cultures take place and shoot germinates either from the callus or
direct. But as in case of somatic embryo the embryo has fate for both root and shoot so this
step is to be followed by acclimatization.

Stage-III (Rooting)
In-vitro grown shoots lack root system. For induction of roots they were transferred to
rooting medium. For rooting half strength MS medium supplemented with auxin is used.

Stage-IV (Acclimatization)
The ultimate success of plant tissue culture depends on the ability to transfer the in vitro
raised plantlet to field or soil conditions with their high survival rate which otherwise will

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lead to high rate of mortality. Because under in vitro condition the high humidity, low light
and poor gaseous exchange in the culture vessels and this allows the plant to grow well but
the natural protective covering of cuticle is not fully developed, they have green leaves but
their chloroplast and other cells are primarily immature in comparison to the field grown
plants and same is true for root system. Thus the direct exposure, from in vitro condition to
the outer atmosphere results in photoinhibition and chlorophyll photobleaching to these
plants results in the high rate of mortality.
Thus the transplantation from completely controlled conditions to the outer environment
should be gradual or step wise and this process of gradually preparing the plants to survive in
the field conditions is called acclimatization. Gradual transfer must include, transferring them
in the paper cups with soil but keeping these plant in controlled condition within the growth
chambers for few days and then only the exposure to low humidity, high light for few hours
to increase a full day before planting in the field.

6.5.2- Organogenesis
Organogenesis is the formation of organs in culture via direct or indirect methods and follows
all above steps (Stage I to Stage IV). Organogenesis is majorly is under the control of growth
regulators and choice of development of particular organs.

6.5.3- Technique in androgenesis

Androgenesis refers to the development of plants (sporophytes) from microspores or
immature pollen (male gametophyte). The basic techniques are same as in case of general in
vitro studies i.e., from stage 0 to stage IV, but this involve the concept of stress in stage 0.
Two techniques are used to produce androgenic haploids, viz. anther culture and isolated
pollen culture.

Anther culture:
Anther culture is one of the simple and efficient methods for haploid production, further this
technique requires minimum facilities. Flower buds, with pollen grains at the most responsive
stage (to be sure that the anthers are at the right stage for culture some of them are selected
anthers are break on glass slide and stain with acetocarmine and examine the anther under
microscope) are surface sterilized (sterilization is generally done with 70 % ethanol and some
time with sodium hypochloride or any other chemicals). Than the anthers excised under
aseptic conditions and culture on semi-solid or in liquid medium. In some cases, where the
flower buds are small, whole buds or inflorescences enclosing the anthers at the appropriate
stage of pollen development are cultured.

Generally it is thought to be put our plant under suitable stress treatment before, the explant
to be collected for culture. Once the calli become visible in the cultures they are further
placed for organogenic or embryogenic differentiation depending upon the objectives on
different medium.

Microspore culture

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Parallel to the anther culture, it is now possible to achieve androgenesis in the cultures of
mechanically isolated pollens. But the special pretreatment is required in some cases further
the plating density is a critical factor for the induction of androgenesis in pollen cultures.
1×104 pollen per ml of the medium is satisfactory.
Further, the application of a stress is necessary to induce microspore embryogenesis. Because
the stress is required to switch the default developmental pathway of microspores toward
embryogenesis and this vary with the species and even within a species. Cold treatment is
preferred stress for further inductions while some time heat treatment or heat shock also used.

6.5.4- adventive embryogenesis

In vitro adventive embryogenesis also know as somatic embryo is one of the popular
technique in plant tissue culture, in this studies the first two stages and fourth stage i.e., stage-
0, stage-I and stage IV remain same as in case of general in vitro studies, but stage- II and
stage III, get merged because in somatic embryo as somatic embryo have fate for both root
and shoots and further they germinate as zygotic embryos to form plants with a primary root
system, so rooting step in not required in this technique.

6.6 Utility
1. A clone is a group of individuals or cells derived from a single parent individual or cell
through asexual reproduction. All the cells in callus or suspension culture are derived
from a single explant by mitotic division. Therefore, all plantlets regenerated from a
callus/suspension culture generally have the same genotype and constitute a clone. These
plantlets are used for rapid Clonal propagation. Besides the calli, nodal explant is also use
for the faster clonal propagation.
2. Genetic variation present among plant cells of a culture is called Somaclonal variation.
Sometime these variations prove to be useful develop such verities which are resistant to
pathogens or stress resistant. Further, some time new mutation may be isolated example
jointless pedicel mutant in tomato.
3. Callus produced via tissue culture is use in secondary metabolite production.
4. The main advantage of haploids in plant breeding programmes is to achieve complete
homozygosity in a single step by doubling the chromosome number.
5. The haploid produced via tissue culture may use for the identification of mutants, since
even recessive mutant alleles if present will expressed in the next generation or in the
regenerated plantlets.
6. Encapsulation of somatic embryo will produce artificial seeds, thus can be store and use
for long time.
7. Production of virus free plants can be raised via plant tissue culture.
8. Seed dormancy, the term means the failure of seeds to germinate although environmental
conditions including water, temperature, light and gases are favorable for germination or
the incapacity of a viable seed to germinate under favorable conditions. So using this
technique seed germination can be foster.
9. Plant tissue culture is used to regenerate plants produced by distant crosses, because
otherwise these may fail due to several reasons.

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10. This technique is useful in many early ripening varieties such as cherry, apricot, plum,
produced seeds are not able to germinate and dies under the soil due to incomplete
embryo development.
11. This technique is used in the production of transgenic plants; a gene of desirable character
is inserted in the cell or transgenes and than the plantlets can be regenerated from these
cells.

6.7 SUMMARY
In present unit, we discuss the different fundamental approaches of plant tissue culture. The
science of plant tissue culture comes with the work of Haberlandt 1902. He himself was
unsuccessful, but subsequent efforts by his successors were also dogged by failures, with the
efforts of many workers and many trials present day this technique became a popular tool in
the field of conservation, gene manipulation, mutant regeneration and many other.
Development of organ from the callus is called as organogenesis. We have two approaches
for the organogenesis in first we can raise our plantlet from explant (node, meristem, bulbs or
embryo) without forming callus and in second we first form callus from any part of the plant
and from the callus we raise plantlets. Besides, organogenesis we may study plant
morphogenesis using this technique, plant morphogenesis corresponds to a biological process
in which the plant assumes its specific form during their development in relation to its
external form and to its internal organization, thus encompassing all levels from the cellular
components until the complete plant is produced. Somatic embryogenesis occurs only under
the controlled environment of plant tissue cultures. Since 1958, when it was reported for the
first time in carrot today somatic embryogenesis is choice of technique for the large scale
plant regeneration, somatic embryo produce plantlet as true as zygotic embryos. Somatic
embryogenesis also serve in the synthesis of artificial seeds. Further, a fascinating outcome of
tissue culture studies initiated at the University of Delhi has been the spectacular
demonstration for the first time of the development of pollen embryoids and plantlets from
anther culture of Datura by Guha and Maheshwari. Their work stumbled into the discovery
which revolutionizes plant breeding programmes of the future world. Anther culture is a
technique of culturing anther/pollen under aseptic in vitro condition to raise haploid plantlets.
Plants breeders are interested in haploid plants because either spontaneous doubling or an
application of the chemical colchicines to double the chromosome number gives rise to
homozygous plant. The main factors that hinder the application of anther and pollen culture
to cereals are low rates of androgenesis and the high frequency of albinism.

6.8 GLOSSARY
Anther culture: Production of haploid plant by culturing anther on suitable culture medium.
Artificial Seeds: A gel bed containing somatic embryo shoot bud, necessary nutrient and
growth regulators etc, needed for the development of complete plantlet.
Auxins: A class of growth regulators, that are primarily associated with enhancing cell
division, cell elongation and root initiations in cultured cells. IAA, IBA, NAA and 2,4-D are
some commonly used auxins in Plant tissue cultures.
Caulogenesis: Shoot regeneration from the cultured cells or stem organogenesis.

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Colchicine: An alkaloid from the bulb of Colchicum autumnale that interferes with the
formation of spindle apparatus.
Contaminations: Presence of unwanted cells or micro-organism in an pure or aseptic culture.
Dedifferentiation: Conversion of differentiated plant cells or from specialized organ into
meristematic ones.
Differentiations: Development of different organs from the meristematic cells.
Haploid plants: Plants having half or gametic chromosome number of the species.
Hybrid rescue: Culture of embryo to save hybrid that otherwise die due to degeneration of
endosperm.
Meristem : A group of actively dividing cells from which permanent tissue systems such as
root, shoot, leaf, flower etc are derived
Meristemoid : A group of meristematic cells with in a callus with a potential to form
primordial Embryoid / Somatic embryos : Non zygotic embryo’s formed in culture.
Organogenesis : Type of morphogenesis which results in the formation of organs and / or
origin of shoots roots. The floral organs from tissue culture (or) suspension culture
Rhizogenesis: Root regeneration from the cultured cells in plant tissue culture.
Sub culture : Aseptic transfer of a part of a culture to a fresh medium.
Totipotency : The ability inherent property of a cell (or) tissue to give rise to whole plant
irrespective of their ploidy level and the form of specialization

6.9 SELF ASSESSMENT QUESTIONS

6.9.1-Short notes
Q1. Define organogenesis.
Q2. What is morphogenesis?
Q3. Write a short note on adventive embryogenesis.
Q4. Differentiate between direct organogenesis and in-direct organogenesis.
Q5. Note on factor affecting androgenesis.
Q6. Define explant and its importance.

6.9.2- Multiple choice questions

Q1.The experimental plant piece subjected to tissue culture is referred to as
(a) In vitro culture (c) Explant
(b) Nurse tissue (d) Callus
Q2. Artificial seeds are
(a) seeds produced in laboratory condition
(b) seeds encapsulated in a a gel
(c) somatic embryos encapsulated in a gel
(d) zygotic embryos encapsulated in a gel
Q3. The pair of hormones required for a callus to differentiate
(a) Ethylene and Auxin (b) Auxin and cytokinin
(c) Auxin and Abscisic acid (d) Cytokinin and gibberellin
Q4. Totipotency refers to

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(a) capacity to generate genetically identical plants

(b) capacity to generate a whole plant from any plant cell / explant.
(c) capacity to generate hybrid protoplasts.
(d) recovery of healthy plants from diseased plants.
Q5. Androgenic haploid were first produced by
(a) Guha and Maheshwari (b) Maheshwari and Johri
(c) Maheshwari and White (d) Rangaswamy
Q6. What is an explant?
(a) A part of plant grown under soil
(b) Any part of a plant taken out and grown in a test tube
(c) A specific part of a plant grown in a test tube
(d) Leaves grew under test tube
Q7. What is Callus?
(a) Tissues that grow to form an embryoid
(b) An unorganised actively dividing the mass of cells maintained in a culture
(c) An insoluble carbohydrate
(d) A tissue that grows from an embryo
Q8. Haploid plants are produced in large numbers by
(a) anther culture (b) Ovary culture
(c) both a and b (d) embryo culture
Q9. Cybrids are
(a) nuclear hybrids
(b) hybrid plants derived from cross pollination
(c) cytoplasmic hybrids
(d) cytological hybrids
Q10. In plant tissue culture, what is term ORGANOGENESIS mean ?
(a) formation of callus culture
(b) formation of root and shoot from callus culture
(c) genesis of plants
(d) none of the above
Answers: 1= c ; 2 = c; 3 = b; 4 =b; 5 =a; 6 = b ; 7 =b ; 8 = c; 9 = c ; 10 = b.

6.10 REFERENCES
Babbar, S.B., Narayan, J.P., Bhojwani, S.S. (2000) Occurrence of albino plants in anther and
pollen cultures: a problem limiting the application of in vitro androgenesis in crop
improvement. Plant Tissue Cult 10:59–87.
Bhojwani, S.S. and Razdan, M.K. (1996) Plant tissue culture: theory and practice, a revised
edition. Elsevier, Amsterdam
Binarova, P., Hause, G., Cenklova, V., Cordewener, J.H.G. and Lookeren, C.M.M. (1997) A
short severe heat shock is required to induce embryogenesis in late bicellular pollen of
Brassica napus L. Sex Plant Reprod 10:200–2008.
Blakeslee, A.F., Belling, J., Farnham, M.E. and Bergner, A.D. (1922) A haploid mutant in the
Jimson weed Datura stramonium. Science 55:646–647.

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Guha, S. and Maheshwari, S.C. (1964) In vitro production of embryos from anthers of
Datura. Nature 204:497.
Johri, B.M. (1982). Experimental Embryology of Vascular Plants. Springer-Verlag, Berlin.
Roberts, E.H., 1972. Loss of Viability and Crop Yield. In: Viability of Seeds, Roberts, E.H.
(Ed.). Chapman and Hall, London, pp: 307-320.
Rolston, M. P. (1978). Water impermeable seed dormancy. The botanical review, 44(3), 365-
396.
Thorpe, T.A. (1981). Plant Tissue culture: Methods and Applications in Agriculture.
Academic Press Inc., New York.

6.11 SUGGESTED READINGS

Bhojwani, S.S. and Dantu, P.K. (2010) Haploid plants. In: Davey MR, Anthony P (eds) Plant
cell culture: essential methods. Wiley-Blackwell, UK
Chawla, H.S. (2006). Introduction to Plant Biotechnology, Oxford & IBH Publishing
Co.Pvt.Ltd. New Delhi.
Narayanaswamy, S. (2000). Plant Cell and Tissue Culture. Tata McGraw-Hill Publishing
Company Limited, New Delhi, India.
6.12 TERMINAL QUESTIONS
Q1. Discuss the factors that may influence anther response and how can anther responses be
increased during culture.
Q2. What function does ABA have in embryo development?
Q3. Explain somatic embryogenesis. Draw the stages of embryogenesis and note when the
different stages were first visible.
Q4. Discuss the anther culture in detail.
Q5. Discuss the morphogenesis and factor affecting morphogenesis.
Q6. Define Haploids. Describe the haploid production via anther culture and their
applications.
Q7. Discuss the utility of haploid culture in horticulture sector.

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UNIT-7-GENETIC ENGINEERING

Contents:
7.1 Objectives
7.2 Introduction
7.3 Genetic Engineering
7.4 Methods of transfer of genes
7.5 Protoplast and Somatic hybridization
7.6 Transgenic
7.7 Development and use of molecular markers in plant breeding
7.8 Summary
7.9 Glossary
7.10 Self Assessment Question
7.11 References
7.12 Suggested Readings
7.13 Terminal Questions

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7.1-OBJECTIVES

After going through this unit learners will be able to answer the following questions.
• What is recombinant DNA technology?
• What are the tools of genetic engineering?
• What is the role of enzymes in recombinant DNA technology?
• What is vector? How can we use the vector for cloning?
• Why different types of vectors required in cloning experiments?
• What do you mean by expression vector? Is it like cloning vector?
• What are different methods of transfer of genes?
• What are protoplasts?
• What is somatic hybridization? What are the advantages of somatic hybridization?
• What is transgenic? What is the need of transgenics? How transgenics produced?
• What is molecular marker? What is the difference between co-dominant and dominant
marker?
• What is polymorphism?
• Why there is a need of molecular marker?

7.2-INTRODUCTION
The concept of the gene as a unit of hereditary information was introduced by the Austrian monk
Gregor Mendel in an 1866 paper entitled ‘Experiments in plant hybridization’. Although Mendel
introduced the concept, the word gene was not used until 25 years after his death. It was coined
by Wilhelm Johansen in 1909 to describe a heritable factor responsible for the transmission and
expression of a given biological trait.
The first evidence as to the physical and functional nature of genes emerged in 1902.
Chromosome theory of inheritance was put forward by William Sutton in 1902. Archibald
Garrod showed that the metabolic disorder alkaptonurea resulted from the failure of a specific
enzyme and could be transmitted in an autosomal recessive fashion in 1902. Hunt Morgan and
colleagues performed the first genetic linkage experiments in 1911 in the fruit fly Drosophila
melanogaster, and hence showed that genes were located on chromosomes and were physically
linked together. In 1942, George Beadle and Edward Tatum found that X-ray induced mutations
in fungi often caused specific biochemical defects, reflecting the absence or malfunction of a
single enzyme. This led to the one gene one enzyme model of gene function. In 1944, Oswald
Avery and colleagues showed that DNA was the genetic material. Thus evolved a simple picture
of the gene– a length of DNA in a chromosome which encoded the information required to
produce a single enzyme.
The definition of recombinant DNA is any artificially created DNA molecule which brings
together DNA sequences that are not usually found together in nature. It is not surprising that the
first cloning experiments were undertaken in E. coli and that this organism became the primary

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cloning host because gene manipulation in this bacterium is technically easier than in any other
organism. Until the mid-1980s, all cloning was cell-based (i.e. the DNA molecule of interest had
to be introduced into E. coli or another host for amplification). In 1983, there was a further mini-
revolution in molecular biology with the invention of the polymerase chain reaction (PCR). This
technique allowed DNA sequences to be amplified in vitro using pure enzymes. The ability to
insert new combinations of genetic material into microbes, animals, and plants offers novel ways
to produce valuable small molecules and proteins; provides the means to produce plants and
animals that are disease resistant, tolerant of harsh environments, and have higher yields of
useful products; and provides new methods to treat and prevent human disease. The alien gene or
foreign gene can be transferred into target host by means of the use of different methods like
direct gene transfer methods and/or vector mediated gene transfer.
As we know totipotency is a characteristic feature of a plant cell and when protoplast is isolated
by removing the cell wall of plasmolysed cell by using different methods, it also shows potential
of regeneration of cell wall, growth and division (Vasil, 1976) under suitable conditions. So it
too can be cultured and regenerated into a whole plant. The absence of cell wall facilitates the
uptake of various organelles including DNA so could be useful in genetic transformation
experiments. Protoplasts can also be useful in the production of hybrid plants (somatic hybrid)
by the fusion of protoplasts which cannot be produced by conventional plant breeding due to
incompatibility. The process of producing somatic hybrids is known as somatic hybridization.
Somatic hybridization is very important for plant breeding as it overcoming common crossing
barriers among plant species and in organelle genetics and breeding so can be used as a tool for
crop improvement.
Different techniques like cloning and PCR amplification are extensively used for number of
purposes in the field of biotechnology including the development and use of DNA based markers
(molecular markers). The molecular markers are based on the polymorphism detected at the level
of small DNA fragments. Till date a broad range of molecular markers are available which are
being used in a variety of ways in plant breeding.

7.3 GENETIC ENGINEERING

Genetic engineering or recombinant DNA technology is a process in which the alteration of the
genetic makeup of cells is done by deliberate and artificial means. Alteration of the genetic
makeup of cells by deliberate and artificial means is possible which could be done by either
adding or replacing or transferring gene(s) to create recombinant DNA in in vitro conditions.
Recombinant DNA can be defined as “DNA molecules constructed outside living cells (in vitro)
by joining two or more natural or synthetic DNA segments of different sources (different
species) to form new DNA molecules that can replicate in a living cell and which would never
occur naturally.”

Basic steps required in genetic engineering or recombinant DNA technology

i) identification of DNA with desirable genes (identifying genes)

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ii) isolation of identified DNA with desirable genes

iii) introduction of identified DNA with desirable genes into the host
iii) maintenance and re-expression of introduced DNA in the host and transfer of the DNA to its
progeny

Uses of genetic engineering or recombinant DNA technology

It can be used to:
• Increase the yield and quality of existing products
• Improve the characteristics of existing products
• Produce existing products by new routes
• Develop novel products not previously found in nature

Tools of genetic engineering

The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the
test tube which, in turn, depends upon the availability of purified enzymes whose activities are
known and can be controlled. So they can therefore be used to make specified changes to the
DNA molecules that are being manipulated. Therefore to manipulate DNA in the test tube, 1) the
availability of purified enzymes is must, 2) activities of purified enzymes should be known and
3) activities of purified enzymes can be controlled. So the purified enzymes can be used to make
specified changes to the DNA molecules that are being manipulated.

Enzymes for DNA manipulation

DNA Polymerases
DNA polymerases are enzymes that synthesize new polynucleotides complementary to an
existing DNA or RNA template. In other words an enzyme that synthesize DNA is known as
DNA polymerase and when it copies an existing DNA or RNA molecules, is known as
template-dependent DNA polymerase.

Mode of action of a template-dependent DNA polymerase

A template-dependent DNA polymerase forms a new polynucleotide whose sequence is dictated,
via the base-pairing rule, by the sequence of nucleotide in the template (DNA or RNA molecule)
that is being copied. The new polynucleotide is always synthesized in 5’ to 3’ direction. New
nucleotide attached at 3’ end with the help of phosphodiester bond.

Important features of template-dependent DNA polymerase

In template-dependent DNA synthesis, DNA polymerase is unable to use an entirely single-
stranded molecule as the template. Why it is so? Answer is because for initiation of DNA
synthesis there must be a short, double- stranded region to provide a 3’ end onto which the
enzyme will add new nucleotide. This short strand is called as primer. So we can say that a
DNA polymerase requires a primer to initiate the synthesis of a new polynucleotide.

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In the test tube, a DNA copying reaction is initiated by attaching to the template a short,
synthetic oligonucleotide, usually about 20 nucleotides in length, which act as a primer for DNA
synthesis. Annealing of the primer to the template depends on complementary base-pairing. The
position within the template molecule at which DNA copying is initiated can be specified by
synthesizing a primer with the appropriate nucleotide sequence. In other words, the primer
determines which part of a DNA molecule is copied.
A second general feature of template-dependent DNA polymerases is that many of these
enzymes are multifunctional, being able to degrade DNA molecules as well as synthesize them.
In addition to 5’to 3’ DNA synthesis capability, DNA polymerases can also have one or both of
the following exonucleases activities.
a) 5’to 3’ exonuclease activity
b) 3’ to 5’ exonuclease activity
a) A 5’to 3’ exonuclease activity is less common, but it possessed by some DNA polymerases.
b) A 3’ to 5’ exonuclease activity enables the enzyme to remove nucleotides from the 3’ end of
the strand that it has just synthesized, if it is incorrect. This is called the proofreading activity
because it allows the polymerase to correct errors by removing a nucleotide that has been
inserted incorrectly.

Types of DNA polymerases

E.coli DNA polymerase I: It is also known as Kornberg polymerase after its discoverer
Arthur Kornberg. This enzyme plays a main role in replication of E.coli genome and possess
both 3’ to 5’ and 5’ to 3’ exonuclease activities. It is unmodified E.coli enzyme and DNA
dependent DNA polymerase.
There is a modified version of Kornberg enzyme, the Klenow polymerase. It was initially
prepared by cutting the natural E.coli DNA polymerase I enzyme into two segments using a
protease. One of these segments retained the polymerase and 3’ to 5’exonuclease activities but
lacked the 5’ to 3’ exonuclease function of the untreated enzyme.
Dear students do you know that an optimum reaction temperature is required for proper
functioning of E.coli DNA polymerase. E.coli DNA polymerase I enzyme has an optimum
reaction temperature of 37°C, a usual temperature of the natural environment of the bacterium,
inside the intestine of mammals such as humans. Therefore , for its proper functioning , the test
tube reactions are incubated at 37 °C and terminated by raising the temperature to 75 °C or
above, destroying its enzymatic activity.

T4 DNA polymerase: T4 is a bacteriophage of E.coli. It’s activities are very much similar to
Klenow fragment of DNA polymerase I. It functions as a 5’ to 3’ DNA polymerase and 3’ to
5’exonuclease but does not have 5’ to 3’ exonuclease activity.
Reverse transcriptase: It is an additional type of DNA polymerase, which is an RNA-
dependent DNA polymerase so makes DNA copies of RNA rather than DNA template. It is
obtained from various retroviruses. This enzyme plays a main role in replication cycle of

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retroviruses, including the human immunodeficiency viruses that causes acquired

immunodeficiency syndrome (AIDS).
In the test tube, a reverse transcriptase (RNA-dependent DNA polymerase) can be used to make
DNA copies of RNA molecules. These copies are called complementary -DNAs (cDNAs).

Taq Polymerase: Taq Polymerase is also known as Thermus aquaticus DNA polymerase I,
obtained from T. aquaticus. T. aquaticus is a thermophilic bacterium which lives in hot springs at
temperature up to 95 °C. Therefore, DNA polymerase I enzyme obtained from it has an optimum
working temperature of 72 °C, hence known as thermostable DNA polymerase. Taq polymerase
is used in PCR.

Nucleases
Nucleases are those enzymes which cleave or cut genetic material (DNA or RNA) by breaking
the phosphodiester bonds that link one nucleotide to the next. Some nucleases are specific for
DNA and some are specific for RNA. Some nucleases works only on double-stranded DNA and
others only on single-stranded DNA. Some are not fussy what they work on.

Types of Nucleases
Nucleases based upon the substrate on which they act are of two types- DNAse or RNAse
Nucleases
(based upon the substrate on which they act)

DNAse RNAse
( which act on or cut the DNA) ( which act on or cut the RNA)

On the basis of position where they act, nucleases are either endonucleases, making cuts at
internal phosphodiester bonds or exonucleases, removing nucleotides from the ends of DNA/or
RNA molecules.
Nucleases
(based upon the position where they act)

Endonucleases Exonucleases

Internal cut nucleotides removed from the ends

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Endonucleases Exonucleases
DNAse which act at a non-specific region in the DNAse which act at the ends or terminal regions of
centre of DNA DNA
Do not require any free DNA ends (i.e. 5’ and 3’ Require a DNA strand with at least two 5’ and 3’
ends) ends
Can act on circular DNA Can not act on circular DNA
They release short segments of DNA They release nucleotide

DNAse which act on specific position or sequences on the DNA are called as restriction
endonucleases and the sequences which are recognized by the restriction endonucleases are
known as restriction sequences or restriction sites or recognition sequences. These sequences are
palindromic. Palindromic sequences are the nucleic acid sequence in a double strand wherein
reading in a certain direction on one strand matches the sequence reading in the same direction
on the complementary strand. Example- 5’-GAATTC-3’
3’-CTTAAG-5’
Thus restriction endonuclease binds to a DNA molecule at a specific sequence and makes a
double stranded cut at or near that sequence. In this way the position of the cuts within a DNA
molecule can be predicted (an important feature of restriction endonuclease beneficial for all
those aspects of recombinant DNA technology in which DNA fragments of known sequence are
required).
The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans, Werner Arber and Hamilton
Smith for the discovery of restriction endonucleases, leading to the development of recombinant DNA
technology. The first practical use of their work was the manipulation of E. coli bacteria to produce
human insulin for diabetics.

Types of restriction endonucleases:

Type I and Type III restriction endonucleases - Restriction sequences and position of cut
may not be same, therefore less useful because the sequence of the resulting fragments are not
precisely known.
Type II restriction endonucleases: Cut is always at the same place either within the
recognition sequence or very close to it. For example- the type II enzyme, EcoRI (isolated from
E.coli) cuts DNA only at the hexanucleotide 5’-GAATTC-3’.
So digestion of DNA with a type II enzyme gives a reproducible set of fragments whose
sequences are predictable if the sequence of the target DNA molecule is known. Therefore they
are very important because of their specificity. These enzymes require Mg++ as cofactor for
cleavage activity and can generate 5’- PO4 and 3’-OH.

The first Type II to be discovered and utilized was EcoRI, which is staggered and its
recognition sequence is 5′-GAATTC-3′ as discussed above.

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When restriction enzymes cut they produce either blunt ends or sticky ends (Figure 7.1). Based
upon the mode of cutting, they are two types. Blunt end cutters and cohesive (sticky) end cutters.

Figure 7.1 –Sticky ends and blunt ends produced by the action of restriction enzyme

1. Blunt end cutters: Blunt end cutters are those Type II restriction enzymes that recognize a
particular recognition site and cut the DNA strand at the same point on both the strand of DNA
within this recognition site. As a result of which the generated DNA strands are completely base
paired. Such fragments are called as blunt ended or flush ended fragments and restriction enzyme
is called as blunt end cutter (Figure7.2).

Figure 7.2- Restriction endonuclease SmaI, a blunt end cutter produced blunt ended fragments

2. Cohesive (sticky) end cutters: Type II restriction enzyme of this class cut the DNA strand
at different points on both the strands of DNA within the recognition sequence. They generated a
short single-stranded unpaired sequence at the end (Figure 7.3). The short single-stranded
sequence is called as sticky or cohesive end because base pairing between the sticky ends stick
the DNA molecule back together again. This cohesive end may contain 5’- PO4 or 3’-OH, based
upon the terminal molecule (also called as 5’ overhangs or 3’ overhangs). Therefore again
classified as 5’end- cutters or 3’end- cutters as the case may be.

Or

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Figure 7.3: Action of sticky end cutters (EcoRI and BamHI) produces sticky ends

Table1: Characteristics of some restriction endonucleases

Restriction Source Recognition sequence and Nature of cut ends

enzyme cleavage site
Eco RI Escherichia coli RY13 5′-G/AATTC-3′ Sticky
3′-CTTAA/G-5′
HindIII Haemophilus influenza Rd 5′-A/AGCTT-3′ Sticky
3′-TTCGA/A-5′
BamHI Bacillus amyloliquefaciens H 5′-G/GATCC-3′ Sticky
3′-CCTAGG/G-5′
HaeIII Haemophilus aegiptius 5′-GG/CC-3′ Blunt
3′-CC/GG-5′
Alu I Arthrobacter luteus 5’-AG/CT-3’ Blunt
3’-TC/GA-5’

The naming of restriction endonucleases: Restriction endonucleases are named in such a

manner that the name provides information about their sources. For this a system was given by
Smith and Nathans (1973) and a simplified version of this is in use today. The key features of
this system are:
• The first letter written in capital letter tells about the genus of the host organism and the
next two letters written in small letters identified species of that particular genus. This
abbreviation is always written in italics.
• Where a particular strain has been the source then this is identified.
• When a particular host strain has several different R-M systems, these are identified by
roman numerals.

Example: EcoRI, In this restriction enzyme E= Genus, co= species, R= Strain, and I= Order of
identification in a bacterium. Table1is showing few characteristics of some restriction enzymes.

Ligases
Ligases are those enzymes which join DNA molecules together by synthesizing phosphodiester
bonds between nucleotides either at the ends of a single molecule or at the ends of two different
molecules as shown below-

One DNA molecule two DNA molecules

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DNA molecule ligated to itself two DNA molecules ligated

DNA fragments that have been generated by treatment with restriction endonuclease can be
joined back together again, or attached to a new partner, by a DNA ligase. Energy is required for
this reation which is provided by adding either adenosine triphosphate (ATP) or nicotinamide
adenine dinucleotide (NAD) to the reaction mixture, depending upon the type of ligase that is
being used.
The most widely used DNA ligase is obtained from E.coli cells infected with T4 bacteriophage.
This enzyme is involved in the replication of Phage DNA and is encoded by the T4 genome. Its
natural role is to synthesize a missing phosphodiester bond between unlinked nucleotides
present in one polynucleotide (one strand) of a double stranded molecule. In order to join
together two restriction fragments in vitro, the ligase has to synthesize two phosphodiester
bonds, one in each strand. So we can say that ligases are the enzymes which seal the nick by
synthesize a phosphodiester bond at this nick

End modification enzymes: End modification enzymes are those enzymes which make
changes to the ends of DNA molecules.
1. Terminal deoxynucleotidyl transferase is one example of end modification enzymes. It
is obtained from calf thymus tissue. It helps in addition of nucleotides one after the other to the
3’ terminus at a blunt end strand, thus modifying a blunt end into sticky end (Homopolymer
tailing). So it is, in fact, known as template independent DNA polymerase because it is able to
synthesize a new DNA polynucleotide without base pairing of the incoming nucleotides to an
existing strand of DNA or RNA.
Homopolymer tailing- Homopolymer tailing is a method of adding similar
nucleotides to the 3 prime end of the DNA strand (blunt end strand ) with
the help of terminal deoxynucleotidyl transferase.

2. Alkaline phosphatase is another example of end modification enzymes. It is obtained from

various sources, including E.coli and calf intestine tissue. It helps in removing phosphate groups
from the 5’ ends of DNA molecules, thus prevents these molecules from being ligated to one
another. Phosphatase which acts in basic buffers with pH 8 or 9 is called as alkaline phosphatase.
Application of alkaline phosphatase treatment is to prevent recircularization of vector plasmid
without insertion of foreign DNA. In this case circularization of the vector can occur only by
insertion of non-phosphatase-treated foreign DNA which provides one 5’terminal phosphate at
each join. One nick at each join remains unligated but after transformation of host bacteria,
cellular repair mechanism reconstitute the intact duplex (Figure 7.4).

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Figure 7.4 –Action of Alkaline phosphatase

3. T4 polynucleotide kinase is also an end modification enzyme. It is obtained from E.coli

cells infected with T4bacteriophage. Function of T4 polynucleotide kinase is just opposite to that
of alkaline phosphatase means it performs the reverse reaction to alkaline phosphatase. It helps
in adding phosphate to 5’ end by transferring the phosphate from ATP to the 5’end of DNA or
RNA.
You can understand the role of different enzymes used in genetic engineering by going through
Table 2.

Vectors
Vectors used in genetic engineering are plasmids, cosmids, lamda phage vectors, shuttle vectors,
BACs and YACs etc.
"Vector" is an agent that can carry a DNA fragment into a host cell. If used for reproducing the
DNA fragment, it is called a cloning vector. If used for expressing certain gene in the DNA
fragment, it is called an expression vector.
For efficient cloning experiment, the same restriction enzyme must be used to cut both the vector
and the DNA sample. Therefore, a vector usually contains a sequence (polylinker) which can
recognize several restriction enzymes so that the vector can be used for cloning a variety of DNA
samples.

Cloning Vectors
Cloning vectors are carrier vehicles used for gene transfer and reproducing the DNA fragment.
Features required to facilitate cloning into a vector are i) Origin of replication, ii) Selectable
marker and iii) Cloning sites. Commonly used vectors for cloning include plasmid, Lambda
phage, cosmid and yeast artificial chromosome (YAC).

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Table 2- Enzymes used in genetic engineering

Enzymes Functions

Removes phosphate groups from 5' ends of DNA (prevents unwanted re-
Alkaline phosphatase
ligation of cut DNA)
Joins compatible ends of DNA fragments (blunt/blunt or complementary
DNA ligase
cohesive ends). Uses ATP

Synthesizes DNA complementary to a DNA template in the 5'-to-

DNA polymerase I
3'direction. Starts from an oligonucleotide primer with a 3' OH end

Digests nucleotides progressiviely from a DNA strand in the 3' -to-5'

Exonuclease
direction
Adds a phosphate group to the 5' end of double- or single-stranded DNA or
Polynucleotide kinase
RNA. Uses ATP

RNase A Nuclease which digests RNA, not DNA

Heat-stable DNA polymerase isolated from a thermostable microbe

Taq DNA polymerase
(Thermus aquaticus)

Vectors based on E.coli plasmids

Plasmids
Extrachromosomal element of DNA in bacteria is termed as plasmid. Term ‘plasmid’ was coined
in 1952 by Lederberg for all extrachromosomal, covalently closed circular (CCC) DNA
molecules. These are small double-stranded, CCC DNA molecules carrying genes for antibiotic
resistance. They do not occur free in nature but are found in a bacterial cell. Plasmids are
naturally occurring, self replicating (replicate independently of the host cell), present in a
bacteria and in the nuclei of some eukaryotic cells. The size of plasmids ranges from a few kb to
near 100 kb. Plasmids in bacteria exist in supercoiled form.

A plasmid can be considered as suitable vector if it posses the following features:

• It can really be isolated from the cell.
• It possesses a replication origin (ORI) sequence (Origin of replication).
• It possesses a single restriction site for one or more restriction enzymes, called cloning
sites. Insertion of a linear molecule at one of these sites does not alter its replication
properties.
• Plasmids contain genes encoding proteins that make the bacteria resistant to antibiotics
such as tetracycline, ampicilline. These are known as selectable markers.
• It can be reintroduced into a bacterial cell.

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• It must have self transfer capacity.

Plasmid’s small size (usually about 3kb) makes them easy to purify from bacterial
cultures, allow the cloned DNA to be recovered easily. Plasmid vectors are ≈1.2–3kb in
size.
Drawbacks:
• A small size and efficiency to transfer maximum of 15 kb of foreign DNA
• Single restriction endonuclease site
• One or more selectable genetic markers

Natural Plasmids: Natural plasmids are those plasmids which occur naturally in bacteria and
are not constructed in vitro for the sole purpose of cloning. Examples- pSC, Col E1 and RSF
2124. In early cloning experiments, the cloning vectors used were natural plasmids such as Col
E1, RSF2124 and pSC101. These plasmids are small and have single site for the common
restriction endonucleases and they have limited genetic markers for selecting transformants.
Figure 7.5A is showing plasmid as a cloning vector.

A B C
Figure 7.5A- Plasmid as cloning vector, B- pBR322, C- pUC 8

For this region, in vitro superior cloning vectors were constructed. In early 1970s many natural
plasmids were artificially modified and constructed as cloning vectors.

pBR322:Plasmid pBR322 was named after its developers Bolivar and Rodriguez in 1977. Here
p stands for plasmid, B for Bolivar, R for Rodriguez and 322 is the number used by them to
designate the plasmid (strain number). Plasmid pBR322 was constructed by ligating restriction
fragments from three naturally occurring E.coli plasmids: RSF2124, pSC101 and Col E1-like
plasmids (Figure 7.5B). It contains the ApR and TcR genes. It has an origin of replication (ori)
that functions only in E. coli. It is maintained at a high copy number in E.coli. Plasmid pBR322
contains 4361 bp.

pBR325: It encodes additional chloramphenicol resistance in addition to ApR and TcR and has a
unique EcoRI site in the CmR gene.

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pBR327: It was derived from pBR322, by deletion of nucleotides between 1427 to 2516. These
nucleotides were deleted to reduce the size of the vector and to eliminate sequences that were
known to interfere with the expression of the cloned DNA in eukaryotic cells. pBR327 still
contains genes for resistance against ampicillin (ApR) and tetracycline (TcR).

pUC: Another cloning vector is pUC where p means plasmid, U means University and C means
California. pUC series was constructed by enhancing the valuable features of pBR322. It was
named so because it was produced at the University of California. So we can say that pUC series
is derived from cloning vector pBR322.
When the pUC plasmid has been used to transform the host cell E. coli the gene in it (lac Z) may
be switched on by adding the inducer IPTG (isopropyl-β-D-thiogalactopyranoside). Its presence
causes the enzyme β-galactosidase to be produced. The functional enzyme is able to hydrolyse a
colourless substance called X-gal (5-bromo-4-chloro-3-indolylb-galactopyranoside) into a blue
insoluble material (5,50-dibromo-4,40–dichloro indigo). However if the gene is disrupted by the
insertion of a foreign fragment of DNA, a non-functional enzyme results which is unable to carry
out hydrolysis of X-gal. This makes the recombinant pUC plasmid to be easily detected as it is
white or colourless in the presence of X-gal (Figure 7.6). An intact non-recombinant pUC
plasmid will be blue since its gene is fully functional and not disrupted. This elegant system,
termed blue/white selection, allows the initial identification of recombinants to be undertaken
very quickly and has been included in a number of subsequent vector systems.

Figure 7.6– Lac selection

So we can say that lac selection or Blue white screening is a method for selection of
recombinants. New recombinant can be identifies by using insertional inactivation. Identifying
recombinant is important because manipulation (process of formation of recombinants) results a
variety of ligation products, including plasmids that have recircularized without insertion of new
DNA.

pUC8: A most popular pUC plasmid is pUC8, a small plasmid having a size of just 2.7 kb.
Along with its origin of replication, it carries two genes -ApR and lac Z' gene. lac Z' gene has a
cluster of restriction sites (Figure 7.5C).

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A gene for ampicillin resistance (ApR)- Presence of this gene in pUC 8 means that a bacterium
having a pUC 8 plasmid, is able to synthesize an enzyme, called β-lactamase. It enables the cell
to withstand the growth inhibitory effect of the antibiotic. Normal E.coli cells (without pUC8
plasmid) are sensitive to ampicillin and can not grow when the antibiotic is present. Ampicillin
resistance is therefore a selectable marker for pUC8.

The lac Z' gene- It codes for a part of the enzyme β-galactosidase (α-peptide portion of β-
galactosidase). β-galactosidase is one of the enzymes involved in the breakdown of lactose to
glucose and galactose. This lac Z' gene in pUC 8, contains a cluster of unique restriction sites.
Ligation of new DNA into any one of these sites results in insertional inactivation of the gene
and hence loss of β-galactosidase activity. This is the key to distinguish a recombinant plasmid
(one that contain an inserted piece of DNA) from a non-recombinant plasmid that has no new
DNA.
[Insertional inactivation- Inactivation of any gene by inserting new DNA somewhere in-
between that gene.]
Screening for β-galactosidase presence or absence is quite easy. The presence of functional β-
galactosidase molecules in the cells is checked by a histochemical test with a compound called
X-gal (5-bromo-4-chloro-3-indolyl- β–D-galactopyranoside). β-galactosidase convert X-gal into
a blue product.
After manipulation, bacteria having pUC8 plasmid allowed to grow in nutrient medium having
agar, ampicillin and X-gal plus an inducer of the enzyme such as isopropyl-β-D-
thiogalactopyranoside (IPTG). Non- recombinant colonies, the cells of which synthesize β-
galactosidase appeared blue in colour. Recombinant colonies with the disrupted lac Z' gene,
unable to make β-galactosidase appeared white (Figure 7.7).

Figure 7.7- Screening for pUC8 recombinants

Image courtesy: Fig 5.10, Gene Cloning & DNA Analysis- An Introduction, T.A.Brown. fifth edition,
blackwell publishing, 2010

This will be clearer by going through Figure 7.8. Plasmids can insert pieces upto 10kb.

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Figure 7.8- Screening for pUC8 recombinants

Cloning vectors based on E.coli bacteriophage genome (Virus based vectors)

Different types of cloning vectors other than plasmids are required. The reason for developing
cloning vectors based on E.coli bacteriophage genome and others was the inability of plasmid
vectors to handle DNA fragments greater than about 10 kb in size. When insert size was large,
either it interfere with the plasmid replication system or rearranged itself in such a way that the
recombinant DNA molecule become lost from the host cell. In addition to this, for preparing a
genomic library for a eukaryote, the cloned fragment should be large enough to contain a whole
gene

Lambda Phage
The first attempt to develop vectors able to handle DNA fragments greater than about 10 kb in
size focused on bacteriophage λ. Lambda (λ) phages are viruses that can infect bacteria. The
major advantage of the λ phage vector is its high transformation efficiency, about 1000 times
more efficient than the plasmid vector. Phage λ genome is 48.5 kb linear double stranded DNA
having ‘sticky end’ (cohesive ends) of about 12 bases at both ends which are complementary in
sequences. These cohesive ends are called ‘cos sites’. Approximately 15 kb of λ genome is
‘optional’ (non-essential region) because it contains genes that are only needed for integration
with the bacterial chromosome (Figure 7.9). So this segment can be deleted without affecting the
ability of phage to infect bacteria for lytic life cycle. Due to the presence of complementary
sequences in cos- sites DNA could adopts a circular structure when it is injected in the host cell
and ligase may seal the gap of the COS sites.
Phage λ genome can be manipulated in the test tube like plasmid. Reintroduction of manipulated
Phage λ genome into E.coli is known as transfection, the term used for uptake of naked phage
DNA. The more efficient uptake system is called ‘in vitro packaging’. DNA cloning using λ
phages as vectors is given in Figure 7.10.

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Figure 7.9- Phage λ genome

Source- human Molecular Genetics, 4ed.Garland Science 2004

The λ phage particle can accommodate upto 52 kb of DNA, so if the genome has 15 kb removed,
then upto 18 kb of new DNA can be cloned. This limit is higher than that for plasmid vector (10
kb).
Wild type λ DNA is not itself suitable as a vector because it’s DNA contains several target sites
for most of the restriction endonucleases, so not itself suitable as a vector. Therefore derivatives
of wild type λ DNA have been developed that either have a single target site at which foreign
DNA can be inserted (insertional vectors) or have a pair of sites defining a fragment that can be
removed (stuffer) and replaced by foreign DNA (replacement vectors).So two basic types of
phage λ vectors have been developed:
1.Insertional vectors
2.Replacement/ substitution vectors.

1. Insertional vectors: In which part or all of the optional DNA has been removed and a
unique restriction site introduced at some position within the trimmed-down genome. They have
a single target site at which foreign DNA can be inserted. Example- λgt 10, λgt 11 and λ ZAP
vectors.

R
λ Insertional vectors

New DNA inserted into the restriction site (R)

λgt 10 and λgt 11 are modified lambda phages designed to clone cDNA fragments.
λgt 10 vector: is a 43 kb double stranded DNA for cloning fragments that are only 7kb in
length.
λgt 11 vector: is a 43.7 kb double stranded DNA for cloning fragments that are less than 6kb in
length
λ ZAP vectors: λ ZAP is a commercially produced cloning vector that includes unique cloning
sites clustered into a multiple cloning site (MCS).

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Figure 7.10- DNA cloning using λ phages as vectors. The DNA to be cloned is first inserted into
the λ DNA, replacing a nonessential region. Then, by an in vitro assembly system the λ virion
carrying the recombinant DNA can be formed.

2. Replacement/substitution vectors: In which the optional DNA is contained within a

stuffer fragment, flanked by a pair of restriction sites that is replaced when the DNA to be cloned
is ligated into the vector.
It means replacement vectors have a pair of sites defining a fragment that can be removed (a
stuffer fragment) and replaced by foreign DNA. Example- λ EMBL3 and λ EMBL4, charon etc.

R R λ Replacement/ substitution vectors.

New DNA replaces the stuffer fragment

EMBL 3 and EMBL4 are used to clone a large fragment (9-25kb) of genomic DNA. These are
used for the construction of genomic libraries. In these, a central non-essential part (about 14kb),
called stuffer can be replaced by a foreign DNA. These two vectors have poly-linkers with
reverse orders of restriction sites with respect to each other (Figure 7.11). They can be used for
preparing genomic libraries in eukaryotes.

Phasmids:
Phasmids are those vectors which have combined properties of phage and plasmid. If phasmid
has M13 ORI region, then it is known as phagemid. So we can say that a phagemid is an

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engineered vector that contains plasmid and M13 components. Phage M13 is a filamentous
phage of E.coli with a single stranded circular DNA genome. The genomes are enclosed in a
protein coat forming a long filamentous form.

Figure 7.11- EMBL3 and EMBL4

Source-Principles of Gene Manipulation and Genomics, S.B.Primrose and R.M.Twyman, 2006

Advantages of Phagemids
Like plasmid vectors, they give high yield of double stranded foreign DNA

Cosmid-based vectors
A cosmid is a plasmid that contains phage sequences that allow the vector to be packaged and
transmitted to bacteria like a phage vector. So cosmid is a plasmid having cos sites. Cosmids can
efficiently clone large pieces of foreign DNA (32-47 kb) so they are attractive vectors for
constructing libraries of eukaryotic genome fragments.
Advantages-It has the following advantages:
• High transformation efficiency, enough to produce a complete genomic library
• The cosmid vector of 5 kb, can carry up to 32-47 kb of foreign DNA whereas plasmid
and λ phage vectors are limited to 10 kb and 25 kb.

Drawback:
Possibility of two or more genome fragments joining together in the ligation reaction, hence
creating a clone containing fragments that were not initially adjacent in the genome. This would
give an incorrect picture of their chromosomal organization.
This problem can be solved by dephosphorylating the foreign DNA fragment so that they cannot
ligate together. This method is very sensitive to the exact ratio of target-to-vector DNAs because
vector-to-vector ligation can occur. Recombinants with duplicated vectors are unstable and break
down in the host.

Cosmids are plasmids that can be packaged into bacteriophage λ particles

Cosmid=cos site+plasmid

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Fosmids:-
Fosmid contain the F plasmid origin of replication and a ʎ cos site. They are similar to cosmids
but have a lower copy number in E. coli, which means that they are less prone to instability
problems.

Cloning Vectors used in eukaryotes

Plasmid vectors are for bacteria, but they are also available for eukaryotic cells (Yeast and
Fungi). Yeast has a natural plasmid, called the 2µm circle.

Yeast artificial chromosome vectors (YACs)

Szastak and Blackbwn (1982) developed the first vector which could be maintained as a linear
molecule, there by mimicking a chromosome known as YACs. It is a high-capacity cloning
system permitting DNA molecules greater than 40-50 kb to be cloned and can amplify large
DNA molecules in a simple genetic background. Artificial chromosomes can be used to clone
large pieces of DNA in yeast
These vectors are propagated in S. cerevisiae, rather than E. coli and are based on chromosomes,
rather than on plasmids or viruses.
The development of YACs was based on the logic that an eukaryotic linear chromosome (Figure
7.12) needs for its replication and stability, not only replication origin, but also the centromere
and the telomere means each chromosome has these three important components.

• The centromere, play a key role during cell division because the centromere sequence helps in
efficient segregation of the chromosomes into the daughter cells by attaching to the mitotic
spindle during cell division
• The telomere, special sequences that mark the ends of chromosomal DNA molecules preserve
the integrity of the ends of the linear chromosome.
• One or more origin of replication, which initiate synthesis of new DNA when the chromosome
divides.

Ones these elements were provided, the vector could replicate stably like a chromosome and
could accommodate chromosomes sized inserts.

Figure 7.12- Chromosome structure

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In YACs, the DNA sequences that underlies these chromosomal components are linked together
with one or more selectable markers and at least one restriction site into which new DNA can be
inserted.
So we can say that “YACs are artificially constructed linear molecules composed of a
centromere, telomere and replication origin termed an ARS element (autonomous replicating
sequence) which are required for replication and preservation of YACs in yeast cells.” (Figure
7.13)

Figure 7.13- YAC vector pYAC3: a pBR322

YACs are replicated in yeast cell, however the external cell wall of the yeast needs to be
removed to leave a spheroplast.
Standard YACs can accommodate around 600 Kb DNA inserts; special types of YACs can
accommodate upto 1400 Kb DNA inserts. Yeast artificial chromosomes can be used to clone
very large fragments of DNA (50-1800Kb).
Key regions of pYAC vector are:-TEL= yeast telomeres, ARS1= autonomously replicating
sequences, CEN4= centromere from chromosome 4 of yeast, URA3 & TRP 1= yeast maker
genes, Ori= origin of replication of pBR322, YAC vector contain ori compitable in E. coli in
addition to yeast replication origin or an yeast ARS element.

YAC are linear molecules composed of a centromere, telomere & a replication origin termed as
ARS (Autonomous Replicating Sequence). They have restriction enzyme sites and genetic
markers so that they can be traced& selected. 100-2000 Kb of DNA fragments can be inserted in
YACs between centromere & telomere. They can be placed in S. cerevisiae & will be replicated
along with the host chromosome.

Essential components of YAC vectors

• Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for
proliferation in the host cell.
• ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells
containing the YAC vector.
• Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)

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Large insert capacity vectors

BACs and PACs

Other new vector systems for cloning very large DNA inserts in E.coli are the bacterial artificial
chromosomes (BACs) and P1- based artificial chromosomes (PACs). These BACs and PACs are
based on replication control elements from factor F and bacteriophage P1, respectively (Shizuya
et al. 1992; Ioannou et al. 1994).

Bacterial Artificial Chromosomes (BACs):

In order to overcome the difficulties associated with YAC vector, a bacterial cloning system
based on E.coli F factor was designed which was capable of cloning fragments of upto 300-
350kb. These are known as Bacterial Artificial Chromosomes (BACs). BACs are capable of
maintaining human and plant genomic fragments of greater than 300Kb for over 100 generations
with a high degree of stability and have been used to construct genome libraries with an average
insert size of 125 Kb.

• BACs are constructed by using fertility or F factors (present on F plasmid) of E.coli.

• BAC vectors contain ori gene, rep E gene for maintenance of F factor, par A, B, C genes for
plasmid copy number, an antibiotic resistance gene for selection of plasmid, MCS for insertion
of foreign DNA.
• BACs are maintained as single copy plasmids in E.coli.
• Used in genome sequencing projects.
• BAC clones are stable for many generations.

PI derived artificial chromosomes or PACs:-

PACs have combine features of PI vectors and BACs. PACs enable genomic inserts upto 300 Kb
in size to be stably maintained. PACs are vector that can carry much larger fragments of DNA
than cosmids because they do not have packaging constraints.

An expression vector is a special cloning vector that can be used to clone the desired
gene and which also contains the necessary regulatory sequences for obtaining high
level of gene expression.

Table 7.3: Maximum DNA insert possible with different cloning vectors.

Vector Host Insert size

Plasmids E. coli ~10kb
ʎ phage E. coli 5–25 kb
ʎ cosmids E. coli 35–45 kb
P1 phage E. coli 70–100 kb
PACs E. coli 100–300 kb

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BACs E. coli ≤300 kb

YACs S. cerevisiae 200–2000 kb

Expression Vectors
In addition to incorporation of desired gene into host cell, the objective of recombinant DNA
technology is to produce high amount of proteins encoded by the DNA insert. To achieve this
goal, the introduced novel gene must express.
Hence, expression of cloned genes is carried by inserting a promoter sequence (signal for
initiation of transcription) and a ‘terminator sequence’ (that provide signal for termination of
transcription) into the vector. Near cloning site a translation initiation sequence (a ribosome
binding site and short codon) is also incorporated into the vector.
The cloning vectors which contain these signals for protein synthesis are called expression
vector.
In other terms those cloning vectors which in addition to act as vehicles for DNA insert, allow
the DNA insert to be expressed efficiently, are called as expression vectors. Expression vectors
are also known as expression constructs.

Main steps of recombinant technology Artificial Chromosomes (Bacterial or

• Isolation of the genetic material Yeast) have all the elements necessary to
• Cutting of DNA at specific location propagate as a chromosome that can be
• Amplification of gene of interest using PCR used to clone DNA fragments from 100 Kb
• Insertion of recombinant DNA into the host cell/organism to 2000 Kb in length.
• Obtaining the foreign gene product

Applications of recombinant DNA technology

Now recombinant DNA technology has been used in various fields of life and has many
applications like in agriculture, in medicine, in medical diagnosis of diseases, in gene therapy, in
microbial cloning, in cell cloning, in plant cloning as well as in animal cloning.

7.4 METHODS OF TRANSFER OF GENES

Introduction of recombinant DNA molecule carrying desired gene into a suitable host is called
gene transfer. The gene to be transferred in the host is called trans-gene and the organisms that
developed after a successful gene transfer are known as transgenics .There are various methods
of gene transfer or DNA uptake which can be classified into two broad classes (Table 4). They
are (a) Vectorless or direct gene transfer and (b) Vector- mediated gene transfer.
(a) Vectorless or Direct gene transfer or Direct Genetic Transformation
Direct gene transfer has proved to be a simple and effective technique for the introduction of
foreign DNA into the plant genome which takes place without the help of any vector or carrier
vehicle. It has been further sub divided into two subcategories-
1. Physical gene transfer method
2. Chemical gene transfer method

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Physical gene transfer methods

Physical gene transfer includes the following methods for uptake of naked DNA
1. Electroporation
2. Micro injection
3. Biolistics/ particle bombardment/microprojectile
4. Silicon carbide whiskers

Table 7.4-Gene transfer (DNA delivery) methods in plants

Method Salient features
Direct or vectorless DNA transfer
A.Physical methods
Electroporation Mostly confined to protoplasts
Micro injection Limited use since only one cell can be microinjected at a time.
Technical person should be highly skilled
Biolistics/particle bombardment/ Very successful method used for a wide range of plants.
Microprojectile
Liposome-mediated transformation Confined to protoplasts
Silicon carbide whiskers Requires regenerable cell suspensions
B.Chemical methods
Polyethylene glycol mediated method Confined to protoplasts
Calcium phosphate co-precipitation method For the transfection of plant and mostly animal cells
DEAE dextran procedure Does not yield stable trans-formants.
Vector-mediated gene transfer
Agrobacterium mediated gene transfer Very efficient
Plant viral vectors Ineffective hence not widely used

Electroporation
Electroporation is a physical transfection technique for introducing DNA/RNA into plant cells
(protoplast). In this method a precisely controlled brief electrical pulse of high field strength is
used to generate transient, nanometer- sized pores in the cell membrane. For this cells are
exposed to these pulse and reversible/temporary pores are generated. DNA enters the cell
through these pores, and is transported to the nucleus. Process of electroporation can be seen in
figure 7.14. Electroporation has also been successfully applied to rice and sugarcane. Pollen
grains may also be transformed using this process.

Figure 7.14- Process of Electroporation

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Source: BTX, The electroporation experts. btxonline.com

Advantages
• It is a convenient, simple, fast and efficient method applicable to a wide range of plant
protoplasts.
• No toxic side effects on the cells.
Disadvantage
• The most critical parameters are the intensity and duration of the electric pulse, and these
must be determined empirically for different cell types. However, once optimal
electroporation parameters have been established, the method is simple to carry out and
highly reproducible.
• This process has high input cost.
• More number of cells may be required.
• The system requires protoplasts so regeneration protocol is prior requirement so that
these protoplasts regenerate whole plants.
Principle- relatively weak nature of membrane.
A molecule is entered into the host cell through membrane.
Electric pulse is applied which temporarily disturb the phospholipid bilayer, and a compound
enters 10,000 to 1,00,000 v/cm in millisecond or microsecond.

Microinjection
Microinjection is another method of gene transfer. The micro injection technique uses a fine
capillary needle (0.5 to 5.0 micrometer) to deliver DNA precisely into cells in such a way that
the cells are not killed but survive the treatment and can develop further into clones (Figure
7.15).
This procedure is performed under micromanipulator or phase contrast microscope. DNA
transfer via microinjection is suitable in transgenesis, gene knockout study and genetic
engineering.
Advantages
The main advantages of this technique, in addition to being host range independent, are:
(a) The delivery is precise, and can be even into the nuclei of target cells and the amount of DNA
delivered per cells is not limited by the techniques and can be standardised, thus increasing the
chance for integrative transformation.
(b) Small clumps of tissue containing a few cells such a proembryos with high regenerative
potential can be injected.
(c) Desirable genes that are not accessible to Agrobacterium mediated transformation can be
used by this method.
(d) Damage to the cell is less.

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Figure 7.15- Microinjection technique for gene transfer

Source: Biocyclopedia
Disadvantage
Handling requires specialised skill and instrumentations.
Time consuming and laborious.

Particle bombardment/ Microprojectile(Biolistics) gun method

Particle bombardment or biolistics (short term for biological ballistics), is a commonly used
method for genetic transformation of plants and other organisms. In this method particle
bombardment device also known as the gene gun involves coating small metal particles (like
gold, silver, tungsten) with biological molecules such as DNA/RNA and then accelerating them
into target tissues using a powerful force, such as a blast of high-pressure gas, gun powder or an
electric discharge through a water droplet. So millions of DNA-coated metal particles are shot at
target cells or tissues. The DNA elutes off the particles that lodge inside the cells, and a portion
may be stably incorporated in the host chromosomes.The target plant cells or tissues, which are
regeneration competent and accessible to particle penetration, are used for bombardment (Figure
7.16). Microprojectile bombardment has been successfully employed for the transformation of
many plants including a wide range of species such as onion, corn, rice, and wheat, soybean,
cotton, maize, tobacco etc.
Advantages
Easy to use, rapid and versatile.
Small amounts of nucleic acids and few cells are required for efficient transformation
Many cells types can be transfected, including non-dividing cells and plants.

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Figure 7.16- Microprojectile gun method

Source: https://link.springer.com/protocol/10.1385/1-59259-827-7:061

Disadvantages
May cause cell damage.
Requirement of equipment and skill personal is must.
Costly method.

Silicon Carbide
A new method of transforming plant cells is by the use of silicon carbide whiskers/fibres. It was
used first in maize but is now being applied to other crops as well. Silicon carbide
whiskers/fibres are very cheap and are capable of penetrating cells making pores which allow
DNA uptake. The DNA coated silicon carbide fibres are vortexed with plant material (suspension
culture, calluses). During the mixing, DNA adhering to the fibres enters the cells and gets stably
integrated with the host genome. The silicon carbide fibres with the trade name Whiskers are
available in the market.

Advantages
The process is simple and reproducible.
Direct delivery of DNA into intact walled cells. This avoids the protoplast isolation.
Procedure does not involve costly equipment.

Disadvantages
Silicon carbide fibres are carcinogenic and therefore have to be carefully handled.

Chemical gene transfer method

PEG (Polyethylene glycol) mediated gene transfer/ DNA uptake
It involves the use of 15-25% PEG, which stimulates uptake of DNA by endocytosis without any
gross damage to protoplasts. The transformed protoplasts can be selected on a selection medium.

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PEG, in the presence of divalent cations (using Ca2+), destabilizes the plasma membrane of
protoplasts and renders it permeable to naked DNA. In this way, the DNA enters nucleus of the
protoplasts and gets integrated with the genome. The procedure involves the isolation of
protoplasts and their suspension, addition of plasmid DNA, followed by a slow addition of 40%
PEG-4000 (w/v) dissolved in mannitol and calcium nitrate solution. As this mixture is incubated,
protoplasts get transformed.

Advantages:
A large number of protoplasts can be simultaneously transformed.
This technique can be successfully used for a wide range of plant species.

Disadvantages:
The DNA is susceptible for degradation and rearrangement.
Random integration of foreign DNA into genome may result in undesirable traits.
Regeneration of plants from transformed protoplasts is a difficult task.

Calcium phosphate co-precipitation method

The DNA is allowed to mix with calcium chloride solution and isotonic phosphate buffer to form
DNA-calcium phosphate precipitate. When the actively dividing cells in culture are exposed to
this precipitate for several hours, the precipitate is taken up by the cell by the process of
phagocytosis. The recombinant DNA enters the nucleus and integrates into the host’s genome.
the cells get transformed. The success of this method is dependent on the high concentration of
DNA and the protection of the complex precipitate. The transfection efficiency can be increased
by exposing the host cell to 10-20% glycerol or Dimethyl sulfoxide (DMSO). This technique is
used for the transfection of plants and mostly animal cells.

DEAE dextran procedure

The desirable DNA can be complexed with a high molecular weight polymer diethyl amino ethyl
(DEAE) dextran and transferred. The major limitation of this approach is that it does not yield
stable trans-formants.

Liposome mediated transformation or lipofection or liposome transfection

Liposomes interact with DNA spontaneously, fuse with tissue culture cells, and facilitate the
delivery of functional DNA into the cell. This technique is found very successful in the
transfection of plant protoplasts and animal host cells. It is an alternative chemical transfection
procedure to package DNA inside a fusogenic phosopholipid vesicle, which interacts with the
target cell membrane and facilitates DNA uptake (Figure 7.17). This is also known as lipid
mediated transfer. DNA or RNA encapsulated in liposomes can be transferred into plant
protoplast by either direct fusion with the naked plasma membrane or through endocytosis of the
liposome. Although it is efficient in terms of DNA encapsulation and transfer, the method once

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again requires protoplasts and its application is limited to species for which protocols are
available for protoplast regeneration.

Cationic lipid-mediated delivery is a fast, simple, and reproducible means for easily introducing
DNA, RNA, siRNA, or oligonucleotides into eukaryotic cells. It allows the highly
efficient transfection of a broad range of cell types, including adherent, suspension, and insect
cells, as well as primary cultures.

Advantages:
It facilitates transient and stable transformation
The efficiency is also much higher than that of other chemical transfection methods

Disadvantages:
Requires protoplasts.
The major problem with liposome-mediated transformation is the difficulty associated with the
regeneration of plants from transformed protoplasts.

Vector mediated gene transfer

In this method of gene transfer vectors are used. The vector may be a circular double stranded
DNA molecule or RNA molecule. Example- Plasmid of E.coli., Ti plasmid of Agrobactrium,
transposable elements and genome of viruses.

Vectors based on naturally occurring plasmids of Agrobacterium.

Agrobacterium tumefaciens: A plant pathogen, also known as nature’s smallest genetic
engineer. It is gram negative soil bacterium. Gene transfer from bacterium to plant occurs
naturally and causes crown gall disease (the formation of tumors) in plants.
A. rhizogenes: Another species of Agrobacterium is A. rhizogenes which is responsible for
hairy root disease in plants.

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Liposomes can be unilamellar

(20-25 nm), multilamellar (0.1 -
10 µM) vesicles prepared by
sonication of phospholipids and
buffer in organic solvent.
Cationic/neutral lipid mixtures
can spontaneously form stable
complexes with DNA
(lipoplexes) that interact
productively with the cell
membrane, resulting in DNA
uptake by endocytosis (Felgner
et al. 1987, 1994).

Figure 7.17- Lipofection

Both species of Agrobacterium are capable to transfer a particular DNA segment (T-DNA) of the
tumour-inducing (Ti) plasmid/ hairy root inducing (Ri) plasmid into the nucleus of infected cells
where it is stably integrated and transcribed, causing the crown gall disease/hairy root disease,
respectively (Nester et al., 1984; Binns and Thomashaw, 1988).

Ti plasmid
As shown in figure 7.18, Ti plasmid consists of following segments-
T-DNA: It carries two types of genes (1) the oncogenic genes, encoding for enzymes involved
in the synthesis of auxins and cytokinins and responsible for tumor/hairy root formation; (2) the
genes encoding for the synthesis of opines which are produced and excreted by the crown gall/
hairy root cells and act as carbon and nitrogen sources for Agrobacterium.
Left border and right border: The T-DNA fragment is flanked by 25-bp direct repeats
known as border sequences, which act as a cis element signal for the transfer apparatus.
Virulence region: The 30 kb virulence (vir) region is a regulon organised in six operons that
are essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer
efficiency (virC and virE)
The induction of vir gene expression results in the synthesis of proteins that form a conjugative
pilus through which the T-DNA is transferred to the plant cell. Along with these, Ti-plasmid also
contains Opine catabolism and origin of replication regions.

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Figure 7.18- Ti plasmid

So we can say that the Ti-plasmid/Ri plasmid is a natural vector for genetically engineering plant
cells because it can transfer its T-DNA from the bacterium to the plant genome. But wild-type
Ti-plasmids are not suitable as general gene vectors because the T-DNA contains oncogenes that
cause disorganized growth of the recipient plant cells. To be able to regenerate plants efficiently
we must use vectors in which the T-DNA has been disarmed by making it non-oncogenic.
Ti plasmid of A. tumefaciens (causing crown gall disease) and Ri plasmid of A. rhizogenes
(causing hairy root disease) have been used as vectors for gene transfer to plant cells. Ti or
Ri plasmid have two regions called vir region and a T-DNA region. Vir region having six
operons. These are vir A, B, C, D, E and G. Vir region is responsible for virulence towards
host. T-DNA region is transferred to the host, along with the gene intended to be
transferred.

Important points regarding T-DNA

1. The tumour formation is a transformation process of plant cells resulted from transfer
and integration of T-DNA and the subsequent expression of T-DNA genes.
2. The T-DNA genes are transcribed only in plant cells and do not play any role during
the transfer process.
3. The genes on T-DNA are non-essential for transfer and can be replaced with foreign
DNA.
4. Any foreign DNA placed between the T-DNA borders can be transferred to plant cell,
no matter where it comes from.

Ti or Ri plasmid based vectors have been designed with the following properties:
o They do not cause the disease, since they are disarmed by removing disease
causing genes.
o They carry sites for insertion of foreign gene intended to be transferred
o They carry selectable markers (genes which help in selecting the transformed
cells), a powerful promoter (for high level of expression of markers) and a
polyadenylation signal (for adding poly A to mRNA).
A vector having above properties, after insertion of the foreign gene, is transferred to A.
tumefaciens or A. rhizogenes, as the case may be, and then the bacterium is used for infecting the
host cells to which the gene is to be transferred. So we can conclude that during transformation,

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several components of the Ti plasmid enable effective transfer of the genes of interest into the
plant cells. These include:
• T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be
transferred into the plant genome
• Vir genes (virulence genes), which are required for transferring the T-DNA
region to the plant but are not themselves transferred, and
• Modified T-DNA region where the genes that cause crown gall formation are
removed and replaced with the genes of interest.
The first plant transformed by A. tumefaciens was tobacco (Herrera-Estrella, 1983). Figure 7.19
illustrates Agrobacterium-mediated plant transformation and Figure 7.20 represent flow diagram
of it.
The Agrobacterium-mediated transformation process involves a number of steps:
(a) isolation of the genes of interest (target gene) from the source organism;
(b) development of a functional transgenic construct including the gene of interest; promoters to
drive expression; and marker genes to facilitate tracking of the introduced genes in the host
plant;
(c) insertion of the transgene into the Ti-plasmid;
(d) introduction of the T-DNA-containing-plasmid into Agrobacterium;
(e) Host cells (explants) are co-cultivated with Agrobacterium carrying the vector with the
desired foreign gene to allow transfer of T-DNA into plant chromosome.
[Host cell may be any explants like protoplasts, suspension cultured cells, callus cells, leaf discs
microshoots, root segments etc.]
(f) regeneration of the transformed cells into genetically modified (GM) plants; and
(g) testing for transgene expression at lab, greenhouse and field level.

A B

Figure 7.19- A & B, Agrobacterium-mediated gene transfer (Plant Transformation Process)

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Advantages:
Stability of gene transfer is excellent.
Disadvantages:
Has the limitations of host range.

Virus mediated gene transfer (transformation)

Plant viruses can be employed as gene transfer and expression vectors. The vast majority of plant
viruses have RNA genomes. However, the two groups of DNA viruses that are known to infect
plants – the cauliflower mosaic virus (CaMV) and the geminiviruses – were the first to be
developed as vectors because of the ease with which their small, DNA genomes could be
manipulated in plasmid vectors.
Cauliflower Mosaic Virus (Caulimovirus):
The cauliflower mosaic virus (CaMV) is considered as most potential vector for plant
transformation. Its genome consists of a small double -stranded circular DNA of 8 kb length. It
has been used successfully as a vector to allow replication and expression of a foreign gene in
dicotyledonous plants, especially the members of family Brassicaceae. The entire genome has
been cloned in E.coli vector and cloned genome caused infection on turnips. Following infection,
the virus spreads systematically and so does the foreign inserted gene, if any throughout the
plant.
Disadvantage:
No heritable transformation via this vector as neither the gene gets integrated into plant genome
nor the virus transmitted through seeds.

Explants+ Agrobacterium carrying the vector with the desired foreign gene

Co-cultivation to allow infection

Antibiotics to kill bacteria
(cefotaxime)
Transformed and non-transformed tissue

Selective media to kill non-transformed tissue

Transformed tissue/callus

Transformed shoots

Rooting

Adult plantlets

Figure 7.20-Flow diagram of Agrobacterium mediated transformation of explants

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Gemini Virus:
A wheat dwarf virus, a member of gemini viruses, which infects several graminaceous members
has also been cloned and reintroduced into wheat protoplasts.
Viral system usually does not result in stable transformation but they permit high level of
transient expression of desired genes.
In plants the most popular method of transformation (gene transfer) is Agrobacterium mediated
gene transfer, especially in dicotyledonous plants. This is followed by the physical method,
especially particle bombardment in monocotyledonous plants. Chemical methods of gene
transfer require protoplast.

7.5 PROTOPLAST AND SOMATIC HYBRIDIZATION

Protoplast
Hanstein (1880) introduced the term ‘Protoplast’ for living matter enclosed by plant cell
membrane (or plasma membrane). Protoplast can be defined as a plant cell without cell wall. The
term protoplast can also be defined as the spherical plasmolysed content of plant cell enclosed by
plasma membrane.
As we know totipotency is a characteristic feature of a plant cell and when protoplast is isolated
by removing the cell wall of plasmolysed cell by using different methods (will be discussed later
in this unit), it also shows potential of regeneration of cell wall, growth and division (Vasil
1976) under suitable conditions. So it too can be cultured and regenerated into a whole plant. The
absence of cell wall facilitates the uptake of various organelles including DNA. To exploit the
above discussed properties of protoplast, there is a need of isolated protoplasts.

Isolation of Protoplast:
Isolation of protoplast means separation of protoplasts from plant cell/tissue. Plant protoplasts
were first isolated enzymatically by Cocking in 1960 from tomato root cells. Isolation requires
removal of plant cell wall. It can be done by means of two methods.
1. Mechanical method:
In this method cells are plasmolysed and protoplasts are released by cutting the tissue
mechanically with the help of knife. This whole procedure is carried out under the microscope
observation of cells. By this method most of the cells produced broken dead protoplasts. Number
of uncut complete protoplasts is very less. Example- vacuolated cells of storage tissues like
onion bulb scales, tissue of radish root and beet root.
For the first time in 1892, Klercker isolated protoplasts mechanically by micro-surgery of
plasmolysed cells of Stratiotes aloides. Low yield and low viability of protoplast are the main
problems of this method. Besides these it is also very laborious and tedious process.
2. Enzymatic method:
As the name indicates, this method involves the use of enzymes to dissolve the cell wall for
releasing protoplasts. Cocking (1960) obtained protoplasts by degradation of cell wall using
enzymes obtained from a fungus Myrothecium verrucaria (cells were tomato root tips and

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enzyme was cellulase). Later this method become popular with suitable modifications.
Enzymatic method is very easy process in comparison to mechanical method. High protoplast
viability and high yield of protoplasts are the advantages of this method.
Best source for protoplast isolation-Mesophyll tissue from fully expanded leaves of young
plants or new shoots. Why it is so? Answer is- A large number of relatively uniform cells can be
isolated from mesophyll tissue and there is no need to kill the plants.
Enzymes required for digestion of cell wall
Enzymes required for digestion of cell wall should be very effective because we know that cell
wall is made up of cellulose, hemicellulose and pectin. Cellulose, hemicellulose are the
components of primary and secondary structure of cell wall while pectin is a component of
middle lamella that joins the cells. Pectinase degrades the middle lamella while cellulase and
hemicellulase are required to digest the cellulosic and hemicellulosic components of the cell
wall.

Enzymatic method could be used as a one-step method or as a two-step method.

Two-step or sequential method
For isolation of protoplasts enzymes are used one by one. In the first step, the cells are isolated
from callus or tissue by using macerozyme or pectinase that degrade middle lamella and then in
the second step, cellulase is added to this cell suspension to digest the cell wall and release
protoplasts.
One-step or simultaneous method
This method is known as one- step or direct or simultaneous method. The protoplasts are isolated
directly from the tissue by using two enzymes cellulase and pectinase, simultaneously.

Importance of osmoticum
In addition to enzymes, osmoticum is also essential for isolation of protoplasts to prevent the
plasma membrane from rupturing. During enzymatic isolation process an osmoticum is required
which prevents the protoplasts from bursting as the mechanical barrier of cell wall for support is
absent.
Osmoticum
Various osmoticums like ionic substances (potassium chloride or calcium chloride) and non-
ionic substances (mannitol, sorbitol, glucose, fructose, galactose or sucrose) can be used during
enzymatic isolation of protoplasts. For isolation of leaf mesophyll protoplast, mannitol is used as
an effective osmoticum. In general, 0.3 to 0.7M sugar solution can generate suitable osmotic
potential. Generally 50 mM CaCl2 is added to increase the stability of released protoplasts (Rose
1980).

Purification of protoplast:
Pure population of intact and viable protoplasts is required for culture of protoplasts so
purification of protoplasts is must. Purification is done by removing undigested material (debris),
burst protoplasts and enzymes with the help of filtering, centrifugation and washing.

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Protoplast viability
For further use of isolated protoplasts viability check is must which could be done by using
various methods like fluorescein diacetate (FDA) staining method.
Applications:
o Reports about regeneration of complete plants from leaf protoplasts of tobacco by Takebe et
al. (1971) increased the potential of prtoplast culture technique.
o Plant protoplasts can also take up foreign DNA, through their naked plasma membrane,
under specific chemical and physical treatment, so could be useful in genetic transformation
experiments.
o Protoplasts also provide an experimental system for a wide range of biochemical and
molecular studies ranging from investigations into the growth properties of individual cells
to membrane transport.
o Hybrid plants can be produced by the fusion of protoplasts which cannot be produced by
conventional plant breeding due to incompatibility.
o Protoplast can be used to study wall synthesis and deposition.
o Protoplasts can also be studied as single cell systems

Somatic Hybridization
A hybrid produced by fusion of somatic cells of two varieties or species is called somatic hybrids
and the process of producing somatic hybrids is known as somatic hybridization. Development
of hybrid plants through the fusion of isolated somatic protoplasts of two different plant
species/varieties under in vitro conditions is also known as somatic hybridization (Figure 7.21).
Somatic hybridization can also be defined as a technique which allows the manipulation of
cellular genomes by protoplast fusion. Unlike sexual hybridization, the nucleus and cytoplasm of
both parents are fused in the hybrid cells.

When the nucleus of only one parent and cytoplasm of both the parents are fused
in the hybrid cell, then instead of hybrid, it is known as cybrid and process is
known as cybridization.

Plant cells have cell wall so it is very difficult to fuse them. Therefore for somatic hybridization
process some conditions are required.

Requirements for Somatic Hybridization

Somatic cells (without cell wall) from two different parents along with fusogenic agent/
electrofusion method are the main requirements for successful somatic hybridization process. In
addition to this a suitable regeneration system for that desired plant species is also an urgent
requirement.
Basic steps required for somatic hybridization technique
The basic steps required for somatic hybridization technique are following:

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1. Isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and selection of somatic hybrid cells
4. Culture of hybrid cells
5. Regeneration of hybrid plants

Figure 7.21-1- Somatic hybridization. Protoplasts fusion induced by PEG ultimately yields somatic
hybrid cells.2- A diagrammatic representation of protoplast fusion

Fusion of the protoplasts of desired species/varieties

Freshly isolated/prepared protoplasts do not normally fuse together because they are negatively
charged. Presence of negative charge on protoplasts causes repulsion. So the contact of
membrane between the protoplasts is facilitated by the group of chemicals known as fusogens
that compensate for their negative charge.
Chemical fusogens:
PEG (Polyethylene glycol), high concentration of calcium ions (Ca2+) at high pH (8-10) and
elevated temperature (37 °C), NaNO3, Dextran, polyvinyl alcohol, synthetic phospholipids etc.
may be used as fusogens.
Methods of Protoplast Fusion
Fusion of protoplasts can be achieved by applying either spontaneous fusion method or induced
fusion method.
1. Spontaneous Fusion Method:
During enzymatic degradation of cell wall some of the adjacent protoplasts may fuse together to
form homokaryons, sometimes more than two protoplasts fuse together and form multinucleate
cells. Spontaneous fusion method is not in very much use because the spontaneous fusion
products do not regenerate into whole plants except for undergoing a few divisions.
2. Induced Fusion Method:

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Freshly isolated protoplasts can be induced to undergo fusion, irrespective of their origin. This is
done by applying different kinds of fusogen (fusion inducing agents), as the case may be, during
the process.
a. Sodium nitrate (NaNO3) Treatment:
It was for the first time reported by Power et al. (1970). In this method NaNO3 is used as a
fusogen. Isolated protoplasts are suspended in a mixture of 5.5% NaNO3 and 10% sucrose
solution and incubated in a water bath at 35°C for 5 min and centrifuged for 5 min at 200 x g.
Following centrifugation the supernatant is decanted and the pellet is incubated in a water bath at
30°C for 30 min. During this period most of the protoplasts undergo cell fusion. After washing
with osmoticum they are plated properly in culture medium for further growth.
b. High Calcium Ions at High pH:
Keller and Melcher (1973) found this method suitable for protoplasts fusion as positively
charged ions (high concentration of Ca2+) at high pH reduce the net negative charges of
protoplasts. Here the isolated protoplasts are incubated in a solution of osmoticum containing
0.05 M CaCl2 at pH 10.5 (using 50mM glycine-NaOH buffer). Temperature should be 37°C.
Aggregation of protoplasts generally takes place at once and fusion occurs within 10 mins.
c. PEG Treatment:
Polyethylene glycol (PEG) is very effective fusogen. Rate of protoplast fusion is very high while
using PEG as fusogen (Kao and Michayluk, 1974; Wallin et al. 1974). In this process,
protoplasts are suspended in a solution having 28-56% PEG (1500-6000 MW). The tube is then
allowed to settle for 10-40 min at room temperature. After PEG treatment the elution of fusogen
is done by using high pH/Ca++ containing solution which is most effective in enhancing the
fusion frequency. Survival rate of fused protoplasts is also very high in this method.

Electrofusion:
As developed by U. Zimmerman, protoplasts are placed in an electric field and are exposed to
high intensity electric pulse for a short duration (nano to micro second). Due to potential
difference protoplasts line up between the electrodes. An extremely short wave electric shock
reversibly increases permeability of cell membrane and induces protoplast fusion.
In this fusion method, two step procedures is followed: a low voltage and rapidly oscillating AC
field is applied, which causes the protoplasts to become aligned into chains by cell to cell
contact. Second step is brief application of a high voltage DC pulse which induces reversible
breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent
membrane reorganisation. Heterokaryons produced by this electro-fusion divide normally in
culture medium and have the capability of regenerating the plantlet. Yield of somatic hybrids by
this method is very high. Fusion process is synchronous and extends over a short duration so the
hybrids do not lose the viability.
There are different methods of protoplasts fusion but generally three methods are in frequent use
for protoplasts fusion. These are (a) high Ca++ and high pH; (b) Polyethylene glycol (PEG) and
(c) electric field.

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Mechanism of Protoplast Fusion:

You can understand the mechanism of protoplast’s fusion by going through the following three
steps:
(i) Agglutination or adhesion:
When the protoplasts are treated with fusogen (any type), they come in close proximity and their
membranes agglutinated.
(ii) Fusion of Plasma Membrane at Localised Sites:
As soon as the protoplasts come in close contact, specially at the point of adhesion. There is a
formation of cytoplasmic bridges between the protoplasts. The high pH and high Ca++ ions have
shown to neutralize the normal surface charge so that the agglutinated protoplasts can fuse due to
intermingling of lipid molecules in membranes.
(iii) Formation of Heterokaryon:
Rounding off of the fused protoplasts occur due to the expansion of cytoplasmic bridges. This
ultimately results in spherical heterokaryon or homokaryon formation. However some of the
protoplasts remain unfused (Figure 7.22).

Figure 7.22– Possibilities of fusion

Identification and selection of hybrid cells:

Following fusion treatment the protoplast population consists of parental type protoplasts,
homokaryotic fused products and also heterokaryotic fusion products (Figure 7.22). After this
step selection of hybrid cells is must. Different methods have been developed for the selection of
somatic hybrids. These are visual selection, selection by complementation, and/or use of
metabolic inhibitors
Verification and Characterisation of Somatic Hybrid:
Verification of somatic hybrids after regeneration is must. It can be done by clear demonstration
of genetic contribution of both the parents.
(i) Morphological Characters:
Morphological characters are leaf size, leaf surface, pigment, flower shape, pollen character, etc.
In general, the somatic hybrids bear the characters from the parents. Sometimes they have the

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somatic or sexual characters intermediate of both the parents. By going through the
morphological characters somatic hybrids can be verified.
(ii) Isoenzyme Analysis:
Electrophoretic banding patterns of isoenzymes have been extensively used to verify hybridity.
Somatic hybrids can be verified by going through the banding pattern of isoenzyme analysis. In
this method electrophoretic banding pattern is observed. Somatic hybrids may show the banding
pattern of used enzymes like the parents or it may be new type. The enzymes used in this
analysis may be esterase, isoperoxidase, phosphatase, alcohol dehydrogenase, etc.
(iii) Chromosomal constitution:
Counting chromosomes is a reliable and easy method of detection. Cytologically the
chromosome number should be the sum of chromosome number of two parents in somatic
hybrid. Besides the number, the size and structure of the chromosomes of both the parents could
be taken for verification of somatic hybrids.
(iv) Molecular Technique:
Various molecular markers such as RFLP, AFLP, RAPD, microsatellites, etc. are useful for
verification of the somatic hybrids. PCR technology is being utilised for hybrid identification.
Specific restriction patterns of chloroplast and mitochondrial DNA have been used with great
advantage to characterise the somatic hybrids.
Culture and regeneration of the hybrid cells
Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions so
that a complete plant can regenerate from hybrid cells. An outline (Schematic representation) of
protoplast fusion and regeneration of plant is given in Figure 7.23.
Advantages of somatic hybridization
o Its major contribution to plant breeding is in overcoming common crossing barriers
among plant species and in organelle genetics and breeding.
o It can be used as a tool for crop improvement.
o Production of novel interspecific and intergenic hybrids like Pomato (hybrid of potato
and tomato) is possible.
o Production of fertile diploids and polyploids from sexually sterile haploids, triploids and
aneuploids.
o Gene for disease resistance, abiotic stress resistance, herbicide resistance and many other
quality characters can be transferred by it.
o Production of heterozygous lines in the single species is possible by it.
o Production of unique hybrids of nucleus and cytoplasm.
Limitations of Somatic hybridization
o Poor regeneration of hybrid plants.
o Non-viability of fused products.
o Not successful in all plants.
o Production of unfavorable hybrids.
o Lack of an efficient method for selection of hybrids.

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o No confirmation of expression of particular trait in somatic hybrids.

Figure 7.23- Schematic representation of protoplast fusion and regeneration of plant

7.6 TRANSGENICS
Transgenics (plants) are the ones, whose DNA is modified by means of recombinant DNA
technology. Genetic transformation is a method of transferring the gene of interest to the host.
The inserted gene sequence is known as the transgene. It is a key technique for plant molecular
breeding to introduce desirable traits into the existing genomes while preserving
the genetic identity of plants. The inserted genes can come from species within the same
kingdom (plant to plant) or between kingdoms (bacteria to plant). In many cases, the inserted
DNA has to be modified slightly in order to correctly and efficiently express in the host
organism. All these require a laboratory step that is often called gene splicing, or genetic
modification (GM).
There are number of tools or methods which could be applied to achieve the increase in
productivity or change in various quality traits. Efforts have also been made to generate diversity
and produce plants that would not exist in nature. These include use of wide crosses which may
be inter- specific or inter –genetic, use of in- vitro techniques like- embryo rescue so that already
existing variations can be used and protoplast fusion by over coming the species specific barrier
or mutagenesis etc.
To broader the available genetic diversity in a given plant species, the most recent addition in the
long list of methods is generation of transgenic plant by exploiting the techniques of genetic
engineering to transfer a ‘foreign’ DNA into a plant cell.
Cisgenic plants are made up of using genes, found within the same species or a closely related
one, where conventional plant breeding can occur.

Genetically modified organisms (GMO)

Bacteria, fungi, plants and animals whose genes have been altered are called genetically
modified organisms (GMO) or transgenics. Transgenic plants possess a gene or genes that have

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been transferred from a different species. Although DNA of another species can be integrated in
a plant genome by natural processes, the term "transgenic plants" refers to plants created in a
laboratory using recombinant DNA technology. The aim is to design plants with specific
characteristics by artificial insertion of genes from other species or sometimes entirely different
kingdoms.
Food supply in the earth according to demand is still not enough to feed the growing human
population, even after the Green Revolution which succeeded in tripling the food supply.
Increased yields have been due to improved crop varieties, use of agrochemicals (fertilizers and
pesticides). For most of the farmers agrochemicals are too expensive and conventional breeding
is not helping in increasing the yield as per requirements. So there must be an alternative path for
the farmers to obtain maximum yield from their field and to minimize the use of agrochemicals
so their harmful effects on the environment are reduced.
Use of genetically modified crops (GM crops) may be a possible solution. Transgenic plants are
used to express proteins, like the cry toxins from Bacillus thuringiensis, herbicide resistant genes
and antigens for vaccinations.
This process provides advantages like improving shelf life, higher yield, improved quality,
nutritional improvement, disease resistance, insect-pest resistance, herbicide resistance, virus
resistance, tolerant to heat, cold and drought resistance, against a variety of biotic and abiotic
(non-biological) stresses. Transgenic plants can also be produced in such a way that they express
foreign proteins (diagnostic and therapeutic proteins) with industrial and pharmaceutical
value. So we can say that transgenic plants have been deliberately developed for a variety of
reasons as discussed above including edible vaccines. There are two common ways of
introducing DNA into plant cells—indirect, and direct (discussed in gene transfer method
section). Figure 7.24 is showing various steps essential for producing transgenic plants.
Benefits of GM crops
Genetic modification has made
o crops more tolerant to abiotic stresses (cold, drought, heat, salt)
o reduced dependency and reliance on chemical pesticides
o helped to reduce post-harvest losses
o enhanced nutritional value of food e.g., Vitamin A enriched rice.
o increased efficiency of minerals usage by plants (this prevents early exhaustion of fertility
of soil).
First generation transgenic plants- are those transgenic plants which contain only marker genes, that are useful in
the development of transformation systems.
Second generation transgenic plants- are those transgenic plants which in addition to selectable marker genes
contain one or two transgenes, encoding simple agronomic traits such as pest and herbicide resistance.
Third generation transgenic plants-are those transgenic plants that contain multiple transgenes targeting multiple
pests and diseases. These may also express additional value added or agronomic traits.

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Figure 7.24- Steps for generation of transgenic plants

Source:Some Achievements http://www.biology-pages.info/T/TransgenicPlants.html

Some important points

o The first genetically modified crop plant was produced in 1982, an antibiotic-resistant tobacco
plant. The first field trials occurred in France and the USA in 1986, when tobacco plants were
engineered for herbicide resistance.
o The first modern recombinant crop approved for sale in the US, in 1994, was the FlavrSavr tomato,
which was intended to have a longer shelf life.
o The first conventional transgenic cereal created by scientific breeders was actually a hybrid
between wheat and rye in 1876 (Wilson, 1876).
o The first commercially genetically engineered crop marketed in Europe, in 1994, was tobacco
engineered to be resistant to the herbicide bromoxynil, as approved by the European Union
o The country's first pesticide producing crop approved by the US Environmental Protection
Agency, in 1995, was Bt Potato
o The first food with increased nutrient value, in 2000, was Vitamin A-enriched golden rice.
o A regulatory clean line with a β-carotene content of 1.6 µg/g was obtained in 2003.
o In 2004 – Golden Rice meets the sun in the open.
o The first Golden Rice field trial in the world was harvested in September 2004 in Crowley ("where
life is rice and easy"), Louisiana, USA
o Herbicide tolerant soybean, canola, Bt maize, Bt cotton constitute the four major genetically
modified (GM) crops.
o The two commercial transgenic vegetable crops are tomato with delayed fruit ripening and potato
with insect and virus resistance.
o High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein
accumulation.

Some examples of transgenics

Nutritional improvement
Transgenic plants have been developed to improve the nutritional value. For example –
Golden rice: Milled rice is the staple food for a large fraction of the world's human population.
Milling rice removes the husk and any β-carotene it contained. β-carotene is a precursor to
vitamin A. Vitamin A deficiency is widespread, especially in the countries of southeast Asia and
causes half a million children to become partially or totally blind each year.

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The transgenic rice (Golden rice) exhibits an increased production of β-carotene as a precursor
to vitamin A and the seed is yellow in colour. Such yellow or golden rice may be a useful tool to
treat the problem of vitamin A deficiency in young children.
A japonica variety of rice was engineered with three transgenes necessary for the rice grain to
produce and store β-carotene. These included two genes from the daffodil plant and a third from
a bacterium. These three transgenes incorporated into rice that enabled the plants to manufacture
β-carotene in their endosperm. Golden Rice is a new type of rice that contains β-carotene
(provitamin A), which is converted into vitamin A as needed by the body and gives the grain its
golden colour. The original golden rice was called SGR1, and under greenhouse conditions it
produced 1.6 µg/g of carotenoids. Figure 7.25 shows different steps for formation of golden rice.

Figure 7.25 –Steps showing formation of golden rice

1-The genes that give golden rice it’s ability to make β-carotene in its endosperm (the interior of the
kernel) come from daffodils and a bacterium called Erwinia uredovora.
2. These genes, along with promoters (segments of DNA that activate genes), are inserted into plasmids of
Agrobacterium tumifaciens.
3.These agrobacteria are then added to a petridish containing rice embryos. As they “infect” the
embryos, they also transfer the genes that encode the instructions for making β-carotene.
4.The transgenic rice plants must now be crossed with strains of rice that are grown locally and are
suited to a particular region’s climate and growing conditions.
Copyright © 2006 ISAAA.
Source:https://www.isaaa.org/kc/inforesources/biotechcrops/The_Golden_Rice_Technology.htm

Potato is an important food crop. The nutritive value of potato protein is diminished due to
deficiency in essential amino acids lysine, tyrosine and sulphur containing amino acids
methionine and cysteine. To improve the nutritive value of potato an Amaranthus seed albumin
gene AmA I has been expressed in transgenic potato tubers. The protein thus produced is non-
allergenic and rich in all essential amino acids.
High Lysine Corn: Corn is one of the major crops in the world, but its low lysine content is
often problematic for animal consumption. While exogenous lysine supplementation is still the
most common solution for today's feed corn, high-lysine corn has been developed. Reducing the
lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme,

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CordapA, has shown increased total lysine or free lysine content in the grains of modified corn
plants (Huang et al 2005).
Proteins of therapeutic importance, like those used in the treatment, diagnosis of human diseases
can be produced in plants, using recombinant DNA technology. The proteins produced in
transgenic plants for therapeutic use, are of three types – (i) antibodies, (b) proteins and (iii)
vaccines. Antibodies directed against various diseases i.e., dental caries, rheumatoid
arthritis, cholera, E.coli diarrhea, malaria, certain cancers, HIV, rhinovirus, influenza, hepatitis B
virus and herpes simplex virus are known to be produced in transgenic plants. Vaccines against
infectious diseases of the gastro intestinal tract have been produced in plants like potato and
bananas. An anti cancer antibody has recently expressed in rice and wheat seed that recognizes
cells of lung, breast and colon cancer and hence could be useful in both diagnosis and therapy in
the future.
o Golden rice is genetically modified rice that now contains a large amount of Vitamin A.
o More correctly, the rice contains the element β-carotene which is converted in the body into Vitamin-
A. So when you eat golden rice, you get more vitamin A.
o β-carotene gives carrots their orange colour and is the reason why genetically modified rice is golden.
o For the golden rice to make β-carotene three new genes are implanted: two from daffodils and the
third from a bacterium.

Insect resistant plants

Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests. Its lethal
effect is mediated by a protein toxin it produces for which Bt gene is responsible.
Through recombinant DNA methods, its toxin gene can be introduced directly into the genome
of the plant, where it is expressed and provides protection against insect pests of the plant. So
transgene used for insect pest control is Bt gene, isolated from bacteria B. thuringiensis which
produces a toxin protein. When insect larvae ingest these bacteria with their food, protein present
in bacteria kills larvae and this toxic protein is encoded by the Bt gene.
Few strains of B. thuringiensis produce proteins that kill certain insects like lepidopterans
(tobacco budworm, armyworm, butterflies and moths), coleopterans (beetles and weevils),
dipterans (flies, mosquitoes). B. thuringiensis forms protein crystals during a particular phase of
their growth. These crystals contain a toxic insecticidal protein. This Bt toxin (insecticidal)
protein exist as inactive protoxins and when an insect ingest the inactive toxin, it is converted
into an active form of toxin. How this happens? As soon as the inactive toxin comes in the gut
of insect, alkaline pH (7.5-8.0) of the gut and specific digestive proteases solublise the crystals
and inactive form changes into active form. proteases

inactive toxin alkaline pH (7.5-8.0) & active toxin

proteases
This activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell
swelling and lysis and eventually cause death of the insect. But this toxin does not kill the
Bacillus itself. The reason is -Bt toxin protein exist as inactive protoxins and it become active

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only after reaching in alkaline pH of the gut of insect in the presence of specific proteases so
Bacillus remain protected.
Therefore keeping this quality of B. thuringiensis in mind, specific Bt toxin genes (cry) were
isolated from B. thuringiensis and incorporated into the several crop plants like cotton.
Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide
resistance to insects without the need for insecticides.
Transgenic cotton, potato, tomato, corn, tobacco, canola, brinjal have been produced so far
containing Bt gene (insecticidal gene).

Herbicide resistant plants

Plants that can tolerate herbicides are called Herbicide Resistant Plants.
1. Glyphosate tolerant plants- Glyphosate is a broad-spectrum herbicide that controls
broadleaf, sedge, and grass weeds with minimal residual toxicity to crops or non-target
vegetation.
Glyphosate (N-(phosphonomethyl)glycine), commercially introduced in 1974 is a effective
broad spectrum post emergent herbicide and is among the most widely used agricultural
chemicals globally. It is very toxicologically and environmentally safe. Glyphosate is the only
herbicide that targets EPSP (5-enolpyruvyl-shikimate-3-phosphate) synthase enzyme and inhibits
it in an amino acid pathway. So plants die because they lack the key amino acids. Glyphosate is
absorbed across the leaves and stems of plants and is translocated throughout the plant. It
concentrates in meristematic tissue. Initially developed to control the growth of weed species in
agriculture, this herbicide also plays an important role in both modern silviculture and domestic
weed control. The use of this virtually ideal herbicide is now being threatened by the evolution
of glyphosate-resistant weeds. A resistant EPSP synthase gene allows crops to survive spraying
of glyphosate. Glyphosate resistant transgenic tomato, potato, tobacco, cotton, petunia etc. have
been developed by transferring aro A gene into a glyphosate EPSP synthetase from Salmonella
typhimurium and E. coli.
2. Sulphonylurea tolerant plants- Sulphonylurea inhibits acetolactate synthetase (ALS), the
enzyme involved in the biosynthesis of branched chain amino acids. Sulphonylurea
resistant tobacco plants are produced by transforming the mutant ALS (acetolactate synthetase)
gene from Arabidopsis.
3. Atrazine tolerant plants- Atrazine inhibits QB protein of photo system II in chloroplasts.
QB protein of photo system II from mutant Amaranthus hybrids is transferred into tobacco and
other crops to produce atrazine resistant transgenic plants.
4. Bromoxynil tolerant plants- Genes for resistance to some of the newer herbicides have
been introduced into some crop plants and enable them to thrive even when exposed to the weed
killer.

Pest resistant plants

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Several nematodes parasites a wide variety of plants and animals including human beings. For
example a nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a
great reduction in yield. A process of RNA interference (RNAi) was adopted to prevent this
infestation.
Using Agrobacterium vectors, nematode-specific genes were introduces into the host plant. The
introduction of DNA was such that it produced both sense and anti-sense RNA in the host cells.
These two RNA’s being complementary to each other formed a double stranded (dsRNA) that
initiated RNAi and thus, silenced the specific mRNA of the nematode. The consequence was that
the parasite could not survive in a transgenic host expressing specific interfering RNA. So the
transgenic plant got itself protected from the parasite.

Flavr savr
The tomato fruit enzyme polygalacturonase (PG), because of its ability to dissolve cell-wall
pectin, is key to fruit softening. The FLAVR SAVR tomato was developed through the use of
antisense RNA to regulate the expression of the enzyme polygalacturonase (PG) in ripening
tomato fruit. This enzyme as discussed is one of the most abundant proteins in ripe tomato fruit.
So the genetically modified tomato went to U.S. market on May 21, 1994 known as the Flavr
Savr, was no longer able to produce polygalacturonase (PG), due to a deactivated gene.
But high production costs mixed with the company’s inexperience in tomato growing, handling,
and transport led to financial troubles for the Flavr Savr. Eventually Calgene was bought out by
Monsanto, a multinational agricultural company, and the Flavr Savr disappeared from shelves for
good in 1997, just three years after its introduction.

Edible vaccine
Edible vaccines are nothing but transgenic plants that contain agents that trigger an animal’s
immune response. In simple terms, edible vaccines are plant or animal made pharmaceuticals. The
concept of edible vaccines was developed by Arntzen (www.genomenewsnetwork.org) in the
1990s. The genes for proteins to be used in human (and animal) medicine can be inserted into
plants and expressed by them. Corn is the most popular plant for these purposes, but tobacco,
tomatoes, potatoes, rice and carrot cells grown in tissue culture are also being used. The earliest
demonstration of an edible vaccine was the expression of a surface antigen from the bacterium
Streptococcus mutants in tobacco.
Edible vaccines contain DNA fragments from the original pathogen. These fragments code for a
protein that is usually a surface protein of the pathogen. This is responsible for eliciting the
body’s immune response.
Transgenic Potatoes for Diarrhea
The first human trial for an edible vaccine took place in 1997. Volunteers ate transgenic potatoes
that contained the b-subunit of the E. coli heat-labile toxin, which causes diarrhea.
The disadvantage of using potato-based edible vaccine is that it has to be consumed raw; when
cooked the protein may get denatured or in some cases less effective. Research has shown that
by partial boiling at least half the vaccine remained alive.

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Transgenic Banana for Hepatitis B

Genes for protein coat of hepatitis B virus was isolated and introduced into banana. The
transgenic banana is rich in the antigenic protein of hepatitis B virus.
Transgenic Tomatoes against Diarrhea
At Cornell University, US researchers have developed transgenic tomatoes against the norwalk
virus, which causes severe diarrhea. The tomatoes produced a surface protein specific to the
virus. Recently, banana has been explored as an alternative source.
Protein, human growth hormone has been produced by transgenic crop plants with the gene
inserted into the chloroplast DNA of tobacco plants.

Salt Tolerance
A large fraction of the world's irrigated crop land is so laden with salt that it cannot be used to
grow most important crops. However, researchers at the University of California Davis campus
have created transgenic tomatoes that grow well in saline soils. The transgene was a highly-
expressed sodium/proton antiport pump that sequestered excess sodium in the vacuole of leaf
cells. There was no sodium buildup in the fruit.

Virus resistant plants

Genes that provide resistance against plant viruses have been successfully introduced into such
crop plants as tobacco, tomatoes, and potatoes.TMV resistant tobacco and tomato plants are
produced by introducing viral coat proteins. Other viral resistant transgenic plants are Potato
virus resistant potato plants, RSV resistant rice, YMV resistant black gram and YMV
resistant green gram etc.

Terminator Genes
This term is used (by opponents of the practice) for transgenes introduced into crop plants to
make them produce sterile seeds (and thus force the farmer to buy fresh seeds for the following
season rather than saving seeds from the current crop). The process involves introducing three
transgenes into the plant: 1. A gene encoding a toxin which is lethal to developing seeds but not
to mature seeds or the plant. This gene is normally inactive because of a stretch of DNA inserted
between it and its promoter. 2. A gene encoding a recombinase - an enzyme that can remove the
spacer in the toxin gene thus allowing to be expressed. 3. A repressor gene whose protein
product binds to the promoter of the recombinase thus keeping it inactive.
When the seeds are soaked (before their sale) in a solution of tetracycline synthesis of the
repressor is blocked so the recombinase gene becomes active and the spacer is removed from the
toxin gene and it can now be turned on. Because the toxin does not harm the growing plant but
only its developing seeds get affected so the crop can be grown normally except that its seeds are
sterile.

Genetically engineered insulin

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Insulin is a protein hormone produced in the pancreas which has an important function in the
regulation of blood sugar levels. Insulin facilitates the transport of glucose into cells. A
deficiency in insulin is one of the causes of the disease Diabetes mellitus or sugar diabetes
resulting in harmful consequences. D. mellitus, describes a group a metabolic diseases in which
the person has a high blood glucose (blood sugar), either because insulin production is
inadequate or because the body’s cells do not respond properly to insulin or both. Insulin is a
hormone that helps the glucose get into the cells to give them energy.

Diabetes-
Body does not make insulin-type 1 diabetes
Body does not make or use insulin well-type 2 diabetes.
Without enough insulin, the glucose stays in blood.

Insulin consists of two short polypeptide chains: Chain A and chain B, linked together by
disulphide bridges. In mammals, including humans, insulin is synthesized as prohormone which
contains an extra stretch called the C peptide. This prohormone needs to be processed before it
becomes a fully mature and functional hormone. The C peptide is not present in the mature
insulin and is removed during processing.
The main challenge for production of insulin using rDNA techniques was getting insulin
assembled in a mature form. In 1983, Eli Lilly, an American company prepared two DNA
sequences corresponding to A and B chains of human insulin and introduced them in plasmids of
E.coli to produce insulin chains. Chain A and B produced separately, extracted and combined by
creating disulphide bonds to form human insulin (Figure 7.26).

Figure 7.26- Genetically engineered insulin

Recombinant human insulin first entered clinical trials in humans in 1980. At that time, the A
and B chains of the insulin molecule were produced separately and then combined by chemical
techniques. Since 1986, a different recombinant process has been used. The human genetic
coding for proinsulin is inserted into E.coli cells, which are then grown by fermentation to

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produce proinsulin. The connecting peptide is cleaved enzymatically from proinsulin to produce
human insulin (Figure 7.27).
Management of adult-onset diabetes is possible by taking insulin at regular time intervals. Insulin
used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from
animal source, though caused some patients to develop allergy or other types of reactions (being
a foreign protein). Therefore genetically engineered insulin was prepared.

Figure 7.27- Human insulin production

7.7 DEVELOPMENT AND USE OF MOLECULAR MARKERS IN

PLANT BREDD ING
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be
used to identify cells, individuals or species. A marker must be polymorphic, that is, it must exist in
different forms so that chromosome carrying the mutant gene can be distinguished from the
chromosome with normal gene by the form of marker it also carries. This polymorphism in the
marker can be detected at following levels:-
1. Phenotype (morphological)
2. Difference in chromosome characters (cytological)
3. Difference in proteins (biochemical)
4. Difference in the nucleotide sequence of DNA (molecular)
Genetic polymorphism is defined as the simultaneous occurrence of a trait in the same population of
two or more discontinuous variants or genotypes. Morphological, cytological and biochemical
markers are known as Classical markers.
Morphological markers: Those markers which can visually distinguish qualities on the basis of
external appearance (morphology) like seed structure, flower colour, growth habit and other
important agronomic traits, are known as morphological markers.
Cytological markers: Those markers which are related with chromosomes, like- variations present
in their numbers, banding patterns, size, shape, order and position of chromosomes, are known as
cytological markers.

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Biochemical markers: Those markers which are related with biochemical properties (also known
as isozymes). These are multi-molecular forms of enzymes which are coded by various genes, but
have the same functions.
Molecular markers A molecular marker is a DNA sequence that is readily detected and whose
inheritance can easily be monitored. Examples- RFLP, AFLP, SSRs etc.
A molecular marker should have some features:-
1. It must be polymorphic, having ability to detect higher level of polymorphism.
2. Co-dominant inheritance -The different forms of a marker should be detectable in diploid
organism to allow discrimination of homozygous and heterozygous.
3. It should be evenly and frequently distributed throughout the genome.
4. It should be easy to use, fast and cost effective.
5. It should be reproducible.
6. Genetic differences should be clearly visible.
Unfortunately, no single molecular marker meet all these requirements. A wide range of molecular
techniques is available that detect polymorphism at the DNA level. Molecular markers are classified
into various groups:
I. On the basis of mode of gene action molecular markers may be co-dominant or dominant,
means whether markers can discriminate between homozygotes and heterozygotes.
i) co-dominant markers- Those markers which clearly discriminate between homozygotes
and heterozygotes are co-dominant markers or we can say that codominant
markers are markers for which both alleles are expressed when co-occurring in an individual.
Analyze one locus at one time. Example-RFLP, SNPs, CAPS and SSRs.
ii) dominant markers- Those markers which do not tell whether the allele is homozygous or
heterozygous are dominant markers, they only depicts the presence or absence of allele. Allow
for analyzing many loci at one time. Example-RAPD, AFLP, ISSR, SCAR, AP-PCR

Molecular markers

co-dominant markers dominant markers

(RFLP, SNPs, CAPS and SSRs) (RAPD, AFLP, ISSR, AP-PCR)

II. On the basis of mode of method of detection molecular markers may be

i) hybridization-based molecular markers or
ii) polymerase chain reaction (PCR)-based markers
i. Hybridization-based molecular markers

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1. Restriction fragment length polymorphism (RFLP):- RFLP is a non –PCR or hybridization

based marker developed by Grodzicke et al (1974). RFLP was the first technology that enables the
detection of polymorphism at the DNA sequence level. Variation in this DNA sequence is the basis
for the genetic diversity within a species. Principle of RFLP is restriction digestion.

Molecular
markers

Hybridization
based (Non-PCR PCR based
based)

Arbitary/Semiarbi
DNA chip tary Primed Site targeted
RFLP PCR (AFLP)
technology (RAPD, ISSR)

In this method, DNA is digested with restriction enzyme (s), which cut the DNA at any specific
sequences; electrophoresed, blotted on a membrane and probed with a labeled clone. Polymorphism
in the hybridization pattern is revealed and attributed to sequence difference between individuals. The
DNA sequence variation detected by this method was termed RFLP (Botstein et al. 1980) (Figure
7.28).
RFLP variation is environmentally independent. Variation in one DNA fragment obtained with a
specific enzyme is treated as one RFLP. When genomic DNA is digested with one of the restriction
enzymes, a series of fragments are produced of varying length because the recognition sites are not
associated in any type of gene and are distributed randomly throughout the genome. These fragments
upon hybridization yield a characteristic pattern. Variation in this pattern can be caused by base pair
deletion, mutation, inversion or by translocation which result in loss or gain of a recognition site
resulting in a fragment of different length and polymorphism. Genomic restriction fragments of
different lengths between genotypes can be detected on southern blots and by a suitable probe. For
eg.:- Restriction endonuclease Eco R1 cuts DNA at a specific sequence as shown below-

GAATTC G AATTC
CTTAAG CTTAA G

When one or more nucleotides in the endonuclease recognition sequence are altered, EcoR1 will fail
to cut the DNA strand at the altered site and a longer DNA fragment is produced. When a given
restriction enzyme site is present in the DNA molecule of one chromosome but absent from the DNA
molecule of the other homologous chromosome a shorter fragment is produced from the chromosome
with the site, and a longer fragment from the other chromosome. It is now possible to distinguish the
two chromosomes in such an individual on the basis of this RFLP. The individual is heterozygous for
this RFLP and is said to be informative at that locus. Because the RFLP is inherited just like a gene,
one can follow the individual chromosomes as they pass from generation to generation by tracing the
inheritance of the marker fragments.

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So RFLP is a difference in homologous DNA sequences that can be detected by the presence of
fragments of different lengths after digestion of the DNA samples with specific restriction
endonuclease or variation in restriction fragment lengths between individuals of a species when cut
by a restriction enzyme. Most RFLP markers are co-dominant (both alleles in heterozygous sample
will be detected) and highly locus-specific. Probe size:- 0.5-2.0 Kb
The overall method can also be explained by going through Figure 7.29 and as follows:
Step I: Restriction digestion: Extraction of desired fragments of DNA using restriction
endonuclease (RE). The enzyme RE has specific restriction site on the DNA, so it cut DNA into
fragments. Different size fragments are generated along with the specific desired fragments.
Step II: Gel electrophoresis: The digested fragments are run in polyacrylamide gel or Agarose gel to
separate the fragments on the basis of length or size or molecular weight. Fragments of different size
form different bands.
Step III: Denaturation: The gel is placed in sodium hydroxide (NaOH) solution for denaturation to
get single stranded DNA fragments.
Step IV: Blotting: The single stranded DNA fragments obtained are transferred into charge
membrane i.e. Nitrocellulose paper by blotting (called capillary blotting or electro-blotting, as the
case may be).
Step V: Baking and blocking: The nitrocellulose paper having transferred DNA is fixed by
autoclaving. Then the membrane is blocked by using bovine serum albumin or casein to prevent
binding of labelled probe non-specifically to the charged membrane.
Step VI: Hybridization and visualization: The labelled RFLP probe is hybridized with DNA on the
nitrocellulose paper. The RFLP probes are complimentary as well as labelled with radioactive
isotopes so they form colour band under visualization by autoradiography.

Figure 7.28- Procedures of RFLP technique

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Uses of RFLP:-
1. RFLPs are co-dominant markers, enabling heterozygote to be distinguished from homozygote.
2. It permits direct identification of a gene type or cultivar in any tissue at any developmental
stage in an environment independent manner.
3. After identification of gene for particular genetic or hereditary disease, that gene can be
analyzed among other family members
4. It detect mutated gene.
5. It is the basis of DNA finger printing for paternity test, criminal identification etc.(forensic
test).
6. The method is simple as no sequence- specific information is required
Problems:-
1. Labor intensive and expensive.
2. Requires relatively large amount of highly purified DNA.
3. Constant good supplies of probe that can reliably detect variation are needed.
4. Time consuming.
5. Require expertise in autoradiography.

Genomic DNA isolation

Digestion with RE
Fractionation on an agarose gel

DNA in millions of restriction fragments fractionated in the gel by molecular weight

DNA transferred out of the gel on to a membrane filter

Southern hybridization
Auto radiography
RFPL pattern in positive bands
Figure 7.29-Flow diagram of RFLP marker

2. DNA chip technology-

DNA chip technology utilizes microscopic arrays (microarrays) of molecules immobilized on
solid surfaces for biochemical analysis. The microarray technique is based on hybridisation of
nucleic acids. In this technique, sequence complementarity leads to the hybridisation between
two single-stranded nucleic acid molecules, one of which is immobilised on a matrix (Sourthern
et al. 1999). So we can say that in the procedure of genomic analysis, microarrays are exposed to
a labelled sample, hybridised, and complementary sequences are determined (Figure 7.30).

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Figure 7.30-Microarray technology

Source: National Human Genome Research Institute

The basic principal behind DNA chips is the hybridization of samples to immobilized DNA
molecules. There are different names for the microarrays, like DNA/RNA Chips, BioChips or
Gene Chips. The array can be defined as an ordered collection of microspots
(microscopic DNA spots attached to a solid surface), each spot containing a single defined
species of a nucleic acid. As shown in figure 7.31, a DNA chip is a small piece of silicon glass
(~1 cm2) to which a large number of synthetic, single-stranded DNA
oligonucleotides (oligos) have been chemically bonded [left]. Oligos function as DNA probes:
they stick (anneal) selectively only to those DNA molecules whose nucleotide sequences are
exactly complementary: T pairs with A, and G with C. They can therefore be used to identify the
presence of specific DNA sequences in a heterogeneous mixture of genes, for example the
presence of a particular allele against the background of a complete genome. In effect, oligos act
like molecular velcro. A computer reads the pattern of annealing and reports which alleles are
present.

Figure 7.31-Principle of a DNA microarray chip: use as Variant Detector Arrays (VDAs)
(after SM Carr et al. 2008. Comp Biochem Physiol D, 3:11)

DNA chips can be used as Variant Detector Arrays (VDAs) to look for DNA sequences that
differ by single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms (SNPs) are
a type of polymorphism involving variation of a single base pair occurred at the specific
location into the genome. In this example (Figure 7.31), the DNA sequences of
the four oligos highlighted in the first bloc differ only at the last position. To determine which
alleles are present, genomic DNA from an individual is isolated, fragmented, tagged with a
fluorescent dye, and applied to the chip. The genomic DNA fragments anneal only to
those oligos to which they are perfectly complementary. In this case, the allele with
the ~T~ SNP allele binds to the ~~A oligo, and the allele with the ~C~ SNP allele binds to

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the ~~G oligo. A computer reads the position of the two fluorescent tags and identifies the
individual as a C/T heterozygote. [The single spots in the other three columns indicate that the
individual is homozygous at the three corresponding SNP positions].
To determine whether an individual possesses a mutation for a particular disease-A sample of
DNA from the patient's blood as well as a control sample - one that does not contain a mutation
in the gene of interest, are taken. After denaturing the DNA in the samples - a process that
separates the two complementary strands of DNA into single-stranded molecules, manageable
fragments are generated by restriction digestion of the long strands of DNA. Labelling of each
fragment is done by attaching a fluorescent dye. The individual's DNA is labelled with green dye
and the control - or normal - DNA is labelled with red dye. Both sets of labelled DNA are then
inserted into the chip and allowed to hybridize - or bind - to the synthetic DNA on the chip. If the
individual does not have a mutation for the gene, both the red and green samples will bind to the
sequences on the chip that represent the sequence without the mutation (the "normal" sequence).
If the individual does possess a mutation, the individual's DNA will not bind properly to the
DNA sequences on the chip that represent the "normal" sequence but instead will bind to the
sequence on the chip that represents the mutated DNA.
Uses:
o The DNA microarray is a tool used to determine whether the DNA from a particular
individual contains a mutation in genes.
o Scientists use DNA microarrays to measure the expression levels of large numbers of
genes simultaneously or to genotype multiple regions of a genome
o The Gene Chip technology may be employed in diagnostics (mutation detection), in
clinical diagnostic tests for some diseases.
o Microarrays can be used for gene discovery, mapping, polymorphism detection, DNA
resequencing, genotyping on a genomic scale, and gene expression analysis,
Advantages:
High flexibility, which allows creation of a chip with any necessary sequences.
Disadvantages:
DNA chip is simple in concept, but generating probes on a solid array surface requires
considerable expertise and technical tricks.
Time and resources consuming to synthesize the needed number of different oligonucleotides.

ii. PCR based markers:

With the discovery of polymerase chain reaction (PCR) technique, the new generation of PCR-
based DNA markers was developed.
PCR-based markers may be divided into two types:
1. Locus non-specific markers e.g. RAPD, AFLP.
2. Locus specific markers e.g. SSRs, SNPs.
1. Random amplified polymorphic DNA (RAPD):-

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RAPD is a molecular marker based on PCR amplification. RAPD markers are decamer (10
nucleotides long) DNA fragments which are able to differentiate between genetically distinct
individuals, although not necessarily in a reproducible way. Principle of it is DNA amplification.
RAPD used as the first PCR based molecular marker technique developed by Welsh &
Mcckelland (1990) and Williams et al. (1990). RAPD amplification is performed in conditions of
PCR. The DNA isolated from the genome of the species of interest is denatured, the template
molecules are annealed with primers, and amplified by PCR. Single short oligonucleotide
primers (usually a 10-base primer) can be arbitrarily selected to amplify a set of DNA segments
distributed randomly throughout the genome. The amplified products are separated by
electrophoresis and identified. Based on the nucleotide alterations in the genome, the
polymorphisms of amplified DNA sequences differ which can be identified as bands on gel
(Figure 7.32 and 7.33).
Genomic DNA from two different individuals often produces different amplification patterns
(RAPDs). A particular fragment generated for one individual but not for other represents DNA
polymorphism and can be used as a genetic marker. Since each random primer will anneal to a
different region of the DNA, theoretically many different loci can be analyzed. PCR reaction
requires cycling among three temperatures, the first to denature the template DNA strands
(94oC), the second to anneal the primer (35o-40oC) and the third for extension at temperature
optimal for Taq Polymerase (72oC).This cycle is usually repeated 25 to 45 times. Presence of
RAPD band corresponds to a dominant allele, absence of band corresponds to a recessive allele.
Thus heterozygous and homozygous dominant individuals cannot be differentiated with RAPD
markers. DNA polymorphism among individuals can be due to -mismatches at the primer site,
appearance of a new primer site or length of the amplified region between primer sites.
Advantages
o It is fast, simple.
o Relatively inexpensive and easy to perform.
o Need a small amount of DNA (15-25mg).
o Non radioactive assay.
o Does not require species probe libraries
Therefore, RADP gained popularity due to its simplicity, relative ease to perform, non
requirement of prior sequence information and economical in terms of time and money.
Applications
o Construction of genetic maps
o It is useful for assessment of genetic fidelity, hybrid purity, fingerprinting of plant
genomes, cultivar identification and genetic diversity.
o Mapping of traits
o Analysis of the genetic structure of populations
o For identification of somatic hybrids
Disadvantages
• Markers are dominant.

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• Band could not differentiate homozygous or heterozygous individual

3. Amplified fragment length polymorphism (AFLP)

AFLP is a novel technique involving a combination of RFLP and RAPD. The AFLP technique is
based on the selective amplification of subsets of genomic restriction fragments using the PCR
(Figure 7.34). So the principle of it is DNA amplification. Thus, this technique combines the
usefulness of restriction digestion and PCR. Multiple polymorphic markers are simultaneously
produced and can be tested in one PCR. No prior information on genomic DNA sequences is
needed.
Steps:-
1. After isolation genomic DNA is digested with two restriction enzymes to generate
variable genomic DNA fragments.
2. Following heat inactivation of the REs the genomic DNA fragments are ligated to
double- stranded oligonucleotide adaptors to generate template DNA for amplification.
3. Sequences in these adaptors (flanking the genomic DNA fragments) serve as primer
binding sites on the restriction fragments. So it is possible to amplify many DNA
fragments without having prior sequence knowledge.
4. PCR products (Amplicons) are separated on a gel and the resulting band pattern is
visualized by autoradiography.

Figure 7.32- Procedures of RAPD technique

Source: Cultivar identification and traceability, a molecular approach.(2013) by Antonella Pasqualone
In book: Cultivars: chemical properties, antioxidant activities and health benefits. Publisher: Nova
Science Publisher Inc., Editors: K. Carbone

Isolation of DNA
Taq DNA polymerase, primers, dNTPs and buffer in

Denature DNA (94 oC, 1 min)

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Annealing of primer to template DNA strand (35o-40oC, 2 min)

Extension of strand (complementary strand synthesis) (72 oC, 1 min)

25 to 45 cycles

Amplified product separated by gel electrophoresis

Ethidium Bromide stain and seen under UV light

RAPD analysis
Figure 7.33-An outline of RAPD procedure

AFLP
• Highly sensitive method for detecting polymorphism throughout the genome.
• It is a combination of RFLP and RAPD methods.
• Universally applicable and highly reproducible.

Applications: AFLP can be used for mapping, fingerprinting and genetic distance calculation
between genotypes.
Advantages:
• Highly reproducible.
• Can discriminate heterozygote from homozygotes.
• Extremely sensitive method
Disadvantages
• Very expensive.
• Extremely sensitive method.
2. Inter simple sequence repeat (ISSR)
ISSR is another PCR based molecular marker developed by Ziet Kiewwicz et al (1994). ISSR
markers, also popularly known as random amplified microsatellites (RAMs) are present in the
genomes of all eukaryotes. Inter-simple sequence repeats (ISSRs) are regions in the genome
flanked by microsatellite sequences. The term microsatellite was coined by Litt and Lutty
(1989). These are tandemly arranged repeats of mono, di, tri, tetra and penta nucleotides in
different lengths of repeat motifs (eg. A,T,AT,GA,AGG,AAAC etc.). The ISSR marker are
multilocus, mostly dominant genetic markers. Inter-simple sequence repeats (ISSRs) are regions
in the genome flanked by microsatellite sequences.

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Figure 7.34- AFPL method

Source: Mueller and Wolfenbarger (1999)
Here genomic DNA is commonly used as the template for ISSR-PCR (Figure 7.35 A and B). An
ISSR primer is usually 16–25 base pairs (bp) in length, and comprises mainly, or solely, of
repeated DNA motifs (2–4 bp each) meant to be complementary to microsatellite regions in the
genome. ISSR-PCR involves only one primer in each reaction, e.g. single-primer PCR
amplification. However, this single primer actually acts as both the ‘forward’ and ‘reverse’
primers which are essential for an amplification to take place.
Important components of an ISSR experiment:
-Simple sequence repeats, also known as
• gDNA as template for ISSR-PCR
microsatellites.
• ISSR primer design -This term is coined by Litt and Lutty (1989).
• PCR amplification with ISSR primers (ISSR-PCR) -Ideal DNA markers for genetic mapping and
• Gel electrophoresis population studies because of their abundance.

• Scoring of bands
• Estimation of basic parameters

Genomic DNA extraction

Standardise DNA quantity and quality

ISSR primer test,

PCR amplification,
protocol optimization

Amplification of clear bands?

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Yes No
Replicate PCR amplification Exclude primer
with optimised protocol

Bands reproducible?
No Yes
Exclude bands Recruit bands

Genotype population samples

Score recruited bands

Analysis

Figure 7.35A. Recommended process flow chart for ISSR genotyping experiments (Ng and Tan, 2015)

Figure 7.35B- Scoring ISSR bands ( Ng and Tan, 2015)

Applications:
• They are easy to use.
• They are low-cost markers and
• They are methodologically less demanding compared to other dominant markers,
• ISSR is an ideal genetic marker for beginners and for organisms whose genetic
information is lacking.
PCR based approach of microarrays
Microarrays have been widely used as single-nucleotide-polymorphism (SNP) genotyping
platforms. Several alternative approaches have been used to detect SNP’s but the most
commonly used are allele discrimination by hybridization. Allelic discrimination by
hybridization suffers background due to non-specific hybridization in complex genomes. In order
to reduce this background, Affymetrix developed a PCR based approach to reduce genomic
complexity.
In brief, SNPs for their assay are selected to be between restriction sites that are <1kb apart.
Genomic DNA is fragmented with a restriction enzyme, end repaired and adapters for PCR are

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ligated to the fragments. PCR is performed under conditions that selectively amplify products of
<1kb in size. This method reduces genomic complexity by approximately 50-fold and results in a
corresponding increase in signal to noise on the array (Matsuzaki et al., 2004). Today SNP arrays
capable of detecting >1M different human SNPs are available.
Microarrays can be used for expression analysis, polymorphism detection, DNA resequencing,
and genotyping on a genomic scale.

Table 7.5–Comparison of different dominant DNA markers for genetic variation studies
(Ng and Tan, 2015)

7.8 SUMMARY
In this unit we have discussed about the genetic engineering or recombinant DNA technology,
tools used in genetic engineering including different types of enzymes and vectors which are
necessary elements for genetic transformation. This unit includes different methods of transfer of
genes like direct and indirect methods. This section is followed by discussion about protoplast,
isolation and fusion of protoplasts for production of somatic hybrids and somatic hybridization.
After that, unit covers a very important topic i.e., transgenics. It includes introduction of
transgenics and their benefits with examples of transgenics developed so far. Last section of this
unit covers development and use of molecular markers in plant breeding. The whole unit is
summarized in the following key points:
• The basis of recombinant DNA technology is the ability to manipulate DNA molecules in the
test tube
• Enzymes and vectors are main tools of genetic engineering.
• DNA polymerases, are enzymes that synthesize new polynucleotides
• Nucleases are those enzymes which cleave or cut genetic material (DNA or RNA) by
breaking the phosphodiester bonds
• Ligases are those enzymes which join DNA molecules together by synthesizing
phosphodiester bonds between nucleotides

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• Different vectors like plasmids, lambda phage, cosmids, phasmids, BACs, YACs etc are used
as cloning vectors.
• There are various methods of gene transfer for development of transgenics like physical
(electroporation, micro injection, particle bombardment, silicon carbide whiskers), chemical
(PEG mediated, calcium phosphate co-precipitation, DEAE dextran procedure,
lipofection) and Agrobacterium mediated gene transfer,
• Somatic hybridization is a technique which allows the manipulation of cellular genomes by
protoplast fusion.
• Transgenics (plants) are the ones, whose DNA is modified by means of recombinant DNA
technology.
• Transgenic plants have been developed to improve the nutritional value (Goldenrice), to
provide resistance against insects (Bt cotton), herbicides, pests, etc. to increase self life (Flavr
Savr) etc.
• A wide range of molecular markers are available that detect polymorphism at the DNA level
like RAPD, RFLP, AFLP etc.

7.9 GLOSSARY
Cell: Structural and functional unit of life.
Animal cell: An organised unit of protoplasm usually enclosing a central nucleus and
surrounded by a plasma membrane.
Cell: Structural and functional unit of life.
Cloning: Making many identical copies of a cell or DNA molecule or an organism.
Cosmid: A plasmid having cos sites.
E.coli: Rod like bacterium normally found in the colon of humuna and other mammals and
widely used in biomedical research.
Fosmid: vector having the F plasmid origin of replication and a ʎ cos site.
Fusogens: Fusion inducing agents are called fusogens.
Gene: Region of DNA, that controls a discrete hereditary character of an organism.
Hybrid: The progeny (F1, heterozygote) of a cross between two individuals different in one or
more heritable characters (genes).
Hybridization: The mating or crossing of two plants or lines of dissimilar genotypes.
Insertional inactivation- Inactivation of any gene by inserting new DNA somewhere in-
between that gene.
Ligases: Ligases are those enzymes which join DNA molecules together by synthesizing
phosphodiester bonds between nucleotides.
Molecular markers: A DNA sequence that is readily detected and whose inheritance can easily
be monitored.
Nucleases:Nucleases are those enzymes which cleave or cut genetic material (DNA or RNA) by
breaking the phosphodiester bonds that link one nucleotide to the next.
Phasmids: Those vectors which have combined properties of phage and plasmid.

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Plant cell: An organised unit of protoplasm usually enclosing a central nucleus and surrounded
by a plasma membrane outside of which it possess a rigid cell wall.
Primer: A short oligonucleotide that is attached to a single-stranded DNA molecule in order to
provide a start point for strand synthesis.
Protoplast: Naked plant cell having all the components of plant cell excluding the cell wall or
The entire content of the cell bounded by the plasma membrane, except the cellulosic cell wall.
Sexual hybridization: Here male gametes or microspores contribute only haploid nuclear
genome and no cytoplasm while the female gamete contributes both cytoplasm and haploid
nuclear genome.
Shuttle vector- a vector (usually a plasmid) that can propagate in two different host species.
Somatic hybrid: A hybrid produced by fusion of somatic cells of two varieties or species is
called somatic hybrids.
Somatic hybridization: The process of producing somatic hybrids /The technique of hybrid
production through the fusion of isolated somatic protoplasts under in vitro conditions and
subsequent development of their product into a hybrid plant
Totipotency: The potential of plant cell or tissue to develop into an entire plant if suitably
stimulated.
Transgenics: Transgenics (plants) are the ones, whose DNA is modified by means of
recombinant DNA technology.
Vector: An agent that can carry a DNA fragment into a host cell.

7.10- SELF ASSESSMENT QUESTIONS

Short answer type questions
Q. What is Genetic engineering?
Ans. Genetic engineering or recombinant DNA technology is a process in which the alteration of
the genetic makeup of cells is done by deliberate and artificial means.
Q.What is the role of Primer?
Ans.For initiation of DNA synthesis there must be a short, double- stranded region to provide a
3’ end onto which the enzyme will add new nucleotide. This short strand is called as primer. So
we can say that a DNA polymerase requires a primer to initiate the synthesis of a new
polynucleotide.
Q. Name the enzymes necessary for DNA manipulation.
Ans. DNA polymerase, Restriction endonuclease, Ligase, Alkaline phosphatase, etc.
Q. What is Homopolymer tailing?
Ans. Homopolymer tailing is a method of adding similar nucleotides to the 3 prime end of the
DNA strand (blunt end strand ) with the help of terminal deoxynucleotidyl transferase.
Q. Why an optimum reaction temperature is required for proper functioning of E.coli DNA
polymerase I?
Ans. E.coli DNA polymerase I enzyme has an optimum reaction temperature of 37°C, a usual
temperature of the natural environment of the bacterium, inside the intestine of mammals such as

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humans. Therefore , for its proper functioning , the test tube reactions are incubated at 37 °C and
terminated by raising the temperature to 75 °C or above, destroying its enzymatic activity.
Q.What are palindromic sequences?
Ans. The nucleic acid sequence in a double strand wherein reading in a certain direction on one
strand matches the sequence reading in the same direction on the complementary strand.
Example- 5’-GAATTC-3’
3’-CTTAAG-5’
Q.What is Taq DNA polymerase?
Ans. Heat-stable DNA polymerase isolated from a thermostable microbe (Thermus aquaticus).
Q. What is vector?
Ans. Vector is an agent that can carry a DNA fragment into a host cell.
Q. Differentiate between cloning vector and expression vector.
Ans. If vector is used for reproducing the DNA fragment, it is called a cloning vector. If used
for expressing certain gene in the DNA fragment, it is called an expression vector.
Q. Mention the features required to facilitate cloning into a vector.
Ans. Features required to facilitate cloning into a vector are origin of replication, selectable
marker and cloning sites.
Q. What is plasmid?
Ans. Plasmids are naturally occurring, self replicating extra chromosomal material present in a
bacteria and in the nuclei of some eukaryotic cells.
Q. Explain natural plasmids.
Ans. Natural plasmids are those plasmids which occur naturally in bacteria and are not
constructed in vitro for the sole purpose of cloning.
Q. Name few natural plasmids.
Ans. pSC, Col E1 and RSF 2124.
Q. What is the full form of pBR322?
Ans. In plasmid pBR322 p stands for plasmid, B for Bolivar, R for Rodriguez and 322 is the
number used by them to designate the plasmid (strain number).
Q. What is the full form of pUC?
Ans. p means plasmid, U means University and C means California. It was named so because it
was produced at the University of California.
Q. What is lac selection/Blue white screening?
Ans. Lac selection/Blue white screening is a method for selection of recombinants. New
recombinant can be identifies by using insertional inactivation. Identifying recombinant is
important because manipulation (process of formation of recombinants) results a variety of
ligation products, including plasmids that have recircularized without insertion of new DNA.
Q.Why different types of cloning vectors other than plasmids are required?
Ans.The reason for developing cloning vectors based on E.coli bacteriophage genome and others
was the inability of plasmid vectors to handle DNA fragments greater than about 10 kb in size.
When insert size was large, either it interfere with the plasmid replication system or rearranged

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itself in such a way that the recombinant DNA molecule become lost from the host cell. In
addition to this, for preparing a genomic library for a eukaryote, the cloned fragment should be
large enough to contain a whole gene
Q. What is Lambda (λ) phage?
Ans. . Lambda (λ) phage is a viruses that can infect bacteria.
Q. Name the plasmid of eukaryotic cell, Yeast.
Ans.Yeast has a natural plasmid, called the 2µm circle.
Q.Why wild type λ DNA not itself suitable as a vector?
Ans. Wild type λ DNA contains several target sites for most of the restriction endonucleases, so
not itself suitable as a vector.
Q. What is Phage M13?
Ans. Phage M13 is a filamentous phage of E.coli with a single stranded circular DNA genome.
The genomes are enclosed in a protein coat forming a long filamentous form.
Q. Name the components responsible for vector’s stable replication like a chromosome.
Ans. Origin of replication, the centromere and the telomere.
Q.What do you understand by shuttle vector?
Ans. A vector (usually a plasmid) that can propagate in two different host species is known as
shuttle vector. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two
different cell types
Q.What is the insert size of different vectors?
Ans. Insert size for plasmids is ~10kb, for ʎ phage it is 5-25 kb, for ʎ cosmids it is 35–45 kb, for
P1 phage it is 70–100 kb, for PACs it is 100–300 kb, for BACs it is ≤300 kb and for YACs it is
200–2000 kb.
Q. What are direct methods of gene transfer or DNA uptake?
Ans. Physical gene transfer method and chemical gene transfer method
Q. Name the physical gene transfer methods.
Ans. Electroporation, micro injection, biolistics/ particle bombardment/microprojectile, and
silicon carbide whiskers.
Q. What is microprojectile bombardment?
Ans.Shooting foreign DNA into plant cells or tissue at a very high speed. This technique is also
known as particle bombardment, particle gun method, biolistic process, microprojectile
bombardment or particle acceleration.
Q. Name the chemical gene transfer methods.
Ans. PEG (Polyethylene glycol) mediated gene transfer/ DNA uptake, calcium phosphate
co-precipitation method, DEAE dextran procedure and liposome-mediated transformation.
Q.What are fusogens?
Ans: Fusion inducing agents are called fusogens.
Q. What is the other name for indirect methods of gene transfer or DNA uptake?
Ans. Vector mediated gene transfer.
Q. Why wild-type Ti-plasmids are not suitable as general gene vectors?

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Ans.Wild-type Ti-plasmids are not suitable as general gene vectors because the T-DNA contains
oncogenes that cause disorganized growth of the recipient plant cells. To be able to regenerate
plants efficiently we must use vectors in which the T-DNA has been disarmed by making it non-
oncogenic.
Q. Define protoplast.
Ans. Protoplast can be defined as a plant cell without cell wall
Q. Why osmoticum is required in enzymatic isolation of protoplast?
Ans. Osmoticum prevents the plasma membrane from rupturing. During enzymatic isolation
process an osmoticum is required which prevents the protoplasts from bursting as the mechanical
barrier of cell wall for support is absent.
Q. What do you mean by somatic hybridization?
Ans. A hybrid produced by fusion of somatic cells of two varieties or species is called somatic
hybrids and the process of producing somatic hybrids is known as somatic hybridization
Q. What is cybridization?
Ans. When the nucleus of only one parent and cytoplasm of both the parents are fused in the
hybrid cell, then instead of hybrid, it is known as cybrid and process is known as cybridization.
Q. What are transgenics?
Ans. Transgenics (plants) are the ones, whose DNA is modified by means of recombinant DNA
technology.
Q.Why need of Transgenics (genetically modified crops)?
Ans. Food supply in the earth according to demand is still not enough to feed the growing human
population, even after the Green Revolution which succeeded in tripling the food supply.
Increased yields have been due to improved crop varieties, use of agrochemicals (fertilizers and
pesticides). For most of the farmers agrochemicals are too expensive and conventional breeding
is not helping in increasing the yield as per requirements. So there must be an alternative path for
the farmers to obtain maximum yield from their field and to minimize the use of agrochemicals
so their harmful effects on the environment are reduced.
Q. Why golden rice is important?
Ans. The transgenic rice (Golden rice) exhibits an increased production of β-carotene as a
precursor to vitamin A and the seed is yellow in colour. Such yellow or golden rice may be a
useful tool to treat the problem of vitamin A deficiency in young children.
Q. Name the source of genes inserted in golden rice.
Ans. The genes that give golden rice it’s ability to make β-carotene in its endosperm (the interior
of the kernel) come from daffodils and a bacterium called Erwinia uredovora.
Q. Why Bt gene is important?
Ans.When insect larvae ingest B. thuringiensis bacteria with their food, protein present in
bacteria kills larvae and this toxic protein is encoded by the Bt gene.
Q. Why does this toxin not kill the Bacillus?
Ans. Bt toxin protein exist as inactive protoxins and it become active only after reaching in
alkaline pH of the gut of insect in the presence of specific proteases so Bacillus remain protected.

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Q. What is FLAVR SAVR

Ans. It is transgenic tomato. The tomato fruit enzyme polygalacturonase (PG) is responsible for
fruit softening as it is able to dissolve cell-wall pectin. The transgenic tomato due to a
deactivated gene was no longer able to produce polygalacturonase so remain fresh for long time
(increased self life).
Q. What is RNAi?
Ans.A method which involve silencing of a specific mRNA due to a complementary dsRNA
molecule that bind to and prevents translation of the mRNA (silencing). The source of this
complementary RNA could be from an infection by viruses having an RNA genome or mobile
genetic elements (transposons) that replicate via an RNA intermediate.
Q. How do edible vaccines work?
Ans. Edible vaccines contain DNA fragments from the original pathogen. These fragments code
for a protein that is usually a surface protein of the pathogen. This is responsible for eliciting the
body’s immune response.
Q. Why molecular markers are important?
Ans. Because they are able to detect polymorphism at the level of small DNA fragments.
Q. RFLP stand for what?
Ans. Restriction fragment length polymorphism.
Q. What is RAPD?
Ans. Random amplified polymorphic DNA (RAPD) is a molecular marker based on PCR
amplification.

7.11- REFERENCES
Bruening G. and Lyons J.M. (2000) The case of the FLAVR SAVR tomato. California
Agriculture. 54(4): 6-7.
Chaitanya V. K. and Kumar J.U. (2006) Edible Vaccines. Sri Ramachandra Journal of Medicine
1(1):33-34.
Cocking E.C. (1960) A Method for the Isolation of Plant Protoplasts and Vacuoles. Nature, 187,
962-963.
Huang S, Kruger DE, Frizzi A, D'Ordine RL, Florida CA, Adams WR, Brown WE, Luethy
MH. (2005) High-lysine corn produced by the combination of enhanced lysine biosynthesis
and reduced zein accumulation. Plant Biotechnol J. 2005; 3(6): 555-69.
K.C. Sink, R.K. Jain, and J.B. Chowdhury (1992). Somatic Cell Hybridization Kalloo et al.
(eds.), Distant Hybridization of Crop Plants © Springer-Verlag Berlin Heidelberg.
Mohan, K.S. and Manjunath, T.M., 2002. Bt Cotton – India’s First Transgenic Crop. J. Plant
Biol., 29 (3): 225-236.
Mueller, U.G. and Wolfenbarger, L.L.(1999) AFLP genotyping and fingerprinting. Review.
Trends in Ecology and Evolution.14(10): 389-394.
Ng W.L. and Tan S.G. (2015) Inter-Simple Sequence Repeat (ISSR) Markers: Are We Doing It
Right? ASM Sci. J., 9(1), 30–39

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Plant Tissue Culture: Theory and Practice, a Revised edition, Bhojwani S.S. and Razdan M.K.
(2009) North Holland, An imprint of Elsevier, ISBN: 978-81-8147-325-7.
Protoplast culture and somatic hybridization 2010 UK Tomar and PK Dantu. In -Cellular and
Biochemical Sciences Book : https://www.researchgate.net/publication/233540386
Sanford, J. C., Klein, T. M, Wolf, E. D., and Allen, N. (1987). Delivery of substances into cells
and tissues using a particle bombardment process. Particulate Sci. Technol. 5, 27–37.
Southern, E., Mir, K. & Shchepinov, M. (1999) Molecular interactions on microarrays. Nat.
Genet. 21, 5-9.
On line science notes and Biocyclopedia

7.12- SUGGESTED READINGS

• Plant Biotechnology, Hand Book by NIIR Board, Kamla Nagar, New Delhi.pp496.
• Plant Tissue Culture: Theory and Practice, a Revised edition, Bhojwani S.S. and Razdan M.K.
(2009) North Holland, An imprint of Elsevier, ISBN: 978-81-8147-325-7.
• Molecular Biology and Biotechnology- A practical Manual. SD Purohit and Neelu Joshi 2007.
• Introduction to Plant Biotechnology. H.S. Chawla. 2002. Science Publisher Inc. USA.
• Biotechnology and Genomics. P.K. Gupta 2005-2006. Rastogi Publication Merrut, India
250002
• Principles of Gene Manipulation and Genomics, S.B.Primrose and R.M.Twyman, 2006

7.13-TERMINAL QUESTIONS
Q. Name the enzymes used in genetic engineering and discuss about their role in cloning.
Q. What do you mean by cloning vectors? How they are different from expression vectors?
Q. Write an essay about cloning vectors.
Q. What are the different methods of gene transfer for plants? Explain them with their
advantages and disadvantages.
Q. Write in detail about protoplast and somatic hybridization.
Q. What are GMOs? Write about golden rice, Bt cotton, and genetically engineered insulin.
Q. Describe methods of isolation of protoplast.
Q. Explain Somatic hybridization technique.
Q. Discuss electrofusion.
Q. Write an essay on transgenisc.
Q. Describe various types of molecular markers.

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BLOCK-3
BIOSAFETY AND IPR
MOLECULAR BIOLOGY AND BIOTECHNOLOGY (MSCBOT-604)

UNIT-8: BIO- AND FOOD-SAFETY, INTELLECTUAL

PROPERTY RIGHTS AND ETHICAL ISSUES

Contents:

8.1-Objectives
8.2-Introduction
8.3-Bio- and Food-Safety
8.4-Patents
8.5-Trade Secrets
8.6-Copyright
8.7-Trademarks
8.8-Ethical Issues
8.9-Plant Genetic Resources
8.10- Plant varietal protection and registration
8.11- GATT and TRIPS
8.12- Patenting and biological material
8.13- Bio-safety and containment practices
8.14- Food-safety of GM crops
8.15- Summary
8.16- Glossary
8.17- Self Assessment Questions
8.18- References
8.19- Suggested Readings
8.20- Terminal Questions
8.21- Answers to Objective-Type Questions

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8.1-OBJECTIVES
After studying this unit, you will be able to—

• Define Bio- and Food-safety.

• Understand the process of patents and their use.
• Understand and define various terms like Trade Secrets, Copyright, Trademarks etc.
• Know the ethical issues involved.
• List and understand various Plant Genetic Resources.
• Get to know Intellectual Property Rights and understand trade agreements related to them.
• Comprehend the Bio-safety and containment practices and Food-safety of Genetically
Modified (GM) crops.

8.2-INTRODUCTION
Food is the primary need of every organism and human beings are no different. Beside other
necessities, it is paramount. Next comes the Environment in which we all live. With the help
of recombinant DNA technology, scientists have combined DNA sequences from different
sources to create functional DNA molecules with novel properties. These molecules are
introduced in various organisms which may be micro-organisms, plants or certain animals
and such organisms are known as Genetically Modified Organisms (GMOs). The never-
ending demand of food for an ever-increasing population and various factors deteriorating
our efforts to grow more grains have led to various scientific discoveries and inventions
which have further led us to a world engaged in developing high yield Genetically Modified
crops commonly known as GM crops. The development in this regard is a result of our
endeavour to grow more with lesser available resources and get rid of low-quality grains,
vegetables and animal products which are partially eaten by maggots and pests.
Biotechnology is safe when practiced properly. You might have heard of the Genetically
Modified vegetable crops such as Bt Cotton, Bt Brinjal etc. BtBrinjal was developed by
Maharashtra Hybrid Seeds Company (Mahyco), by inserting a gene from the soil Bacterium
Bacillus thuringiensis into the genome of various brinjal cultivars, in order to develop
resistance against lepidopteron insects - the Brinjal Fruit and shoot borer. Brinjal is otherwise
an extremely pest-prone crop and is highly susceptible to Fruit & Shoot Borer (FSB) pest.
But this crop was banned in 2010 following concerns raised on biodiversity and public
health. Hence, besides the various benefits of GM crops, the major concern that strikes our
environmental scientists is the Bio- and Food-safety which is believed to be hampered by this
technology.
In this chapter, we will learn and understand the Bio- and Food-safety concerns that affect
our efforts to develop Genetically Modified Organisms (GMOs) for providing sufficient food
and a healthier environment for a sustainable life on earth.

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8.3 BIO- AND FOOD-SAFETY

8.3.1 Bio-Safety
In the present scenario, where the whole world is under Covid-19 pandemic, you must be
aware of the safe practices to avoid infection from Corona Virus. Such practice, so as to
avoid infections and prevention of pathogens, toxins etc. into our bodies is a small part of the
process of Biosafety. Biosafety refers to “the containment principles, technologies and
practices that are implemented to prevent unintentional exposure to pathogens and toxins, or
their accidental release”.1

With the expansion in the field of genetic engineering and commercialization of transgenic
organisms, scientists have expressed fear about the use of such genetically engineered
microorganisms, transgenic plants and animals that they might disturb the environment:
i. By multiplying rapidly and replacing the native microbes.
ii. By transferring genes related to virulence or pathogenesis into native microbial
populations and increasing their virulence or changing harmless microbes into pathogenic
microbes.

The transgenic plants could pose biological and ecological risks:

i. By the production of toxic or allergic metabolites.
ii. By introducing unexpected new susceptibilities to pathogens.
iii. By transmission of new traits to sexually compatible weeds.
iv. By disturbing ecosystems through persistence or altered reactions to parasites,
symbionts and competitors.
v. By the escape of transgenes providing resistance against antibiotics into other
neighbouring plants.
vi. By providing resistance in insects against pesticides and in weeds against herbicides.

Realizing the possible hazards of cloning recombinant DNA technology, National Institutes
of Health (NIH), USA established the Recombinant Advisory Committee (RAC) in 1974,
which recommended various guidelines from time to time. The United Nations Environment
Programme (UNEP) and World Health Organization (WHO) have also issued guidelines for
safe use of GMOs.

8.3.2 Biosafety Guidelines in India

In India, Ministry of Environment, Forest and Climate Change (MoEF& CC) in 1989, has
laid down rules and procedures for the manufacture, import, use and release of GMOs and
also the products obtained from such organisms. This was done under the Environment
Protection Act (EPA). The Recombinant DNA Advisory Committee (RDAC), Department of
Biotechnology (DBT), New Delhi had laid down Recombinant DNA Safety Guidelines and
Regulations in 1990, which were revised in 1994. While DBT implements the research and
development utilizing GMOs and recombinant products, MoEF& CC implements the large-
scale commercial use of these.

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The essential features of these guidelines and regulations are:

i. Every organization associated with research and development using recombinant

DNA technology has to set up an Institutional Biosafety Committee (IBC). All experiments
concerned with the genetic manipulation in plants and likely to have biohazard potential are
to be reported to IBC.
ii. Review Committee on Genetic Manipulation (RCGM) of DBT is the review
committee which reviews all the approvals of ongoing projects on GMOs. Trial permits are
issued only on the recommendation of RCGM.
iii. For the inspection of monitoring of field experiments, each state has a State
Biotechnology Coordination Committee (SBCC) and a District Level Committee (DLC).
iv. There is a Genetic Engineering Appraisal Committee (GEAC) responsible for the
appraisal of activities involving large scale use of hazardous microorganisms and
recombinants in research and industrial production from the environmental angle. It is also
responsible for appraisal of proposals relating to release of genetically engineered (GE)
organisms and products into the environment including experimental field trials. (REF:
https://geacindia.gov.in/about-geac-india.aspx)
v. The biosafety guidelines recognize three levels of risks in case of experiments with
micro-organisms. These are pathogenicity of micro-organisms, local prevalence of concerned
disease and epidemic causing strains in India.
vi. Experiments with micro-organisms, plants and animals are grouped into following 3
categories—
a. Exempt category for self-cloning experiments.
b. Category requiring intimation of initiation to competent authority i.e. experiments
involving non-pathogenic DNA vector systems.
c. Category requiring review and approval by competent authority i.e. cloning of genes
for toxins, antibiotic resistance etc.
vii. Four biosafety levels (BL-1, BL-2, BL-3 & BL-4) have been recognized, each one for
one particular risk group (RG-1, RG-2, RG-3 & RG-4).
viii. Biological safety in the laboratory is achieved by standard laboratory practices and
containment strategies.
ix. NIH has recommended separate containment facilities for each biosafety level.
x. Biological containment consists of use of vectors and hosts in such a way that it limits
the infective ability of a vector to a specific host and the host-vector survival in the
environment is controlled.
xi. Physical containment aims to limit the spread of dangerous micro-organisms. It
involves good laboratory practices, use of safety equipment and laboratory design.
xii. Experiments with GM plants are carried out under a special environment that is
achieved by using glass house containment.
xiii. To carry out genetic manipulation, permission from the Department of Environment is
needed.
xiv. All products of recombinant DNA technology are subjected to general regulations.
xv. GMOs are to be released under appropriate containment to ensure safety.

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xvi. Planned field experiments with transgenic plants can be carried out only after a
stepwise evaluation inside the green house and data collected for promoter sequence, target
gene sequence, regulatory mechanism, cell line used for shuttling and amplification.

8.3.3 Objectives of Biosafety Guidelines

Biosafety guidelines are developed to ensure protection and regulate research and
development activities using recombinant DNA technology so that the adverse effects of this
technique are minimized while encouraging the research activities. These aim for
i. The safe transfer of ‘insert DNA’ into the vector and of recombinant DNA into the
host organism.
ii. Safe handling and use of living modified organisms resulting from recombinant DNA
technology.
iii. Conservation and sustainable use of biological diversity for using them as a source of
good genes.
iv. Reduce the risk of adverse effects of recombinant DNA on human health.

8.4 FOOD SAFETY

Access to sufficient amounts of safe and nutritious food is key to sustaining life and
promoting good health. Unsafe food containing harmful bacteria, viruses, parasites or
chemical substances can cause more than 200 different diseases – ranging from diarrhoea to
cancers. Around the world, an estimated 600 million – almost 1 in 10 people – fall ill after
eating contaminated food each year.3
Food safety refers to the practice of routines in the preparation, handling and storage of food
so as to prevent foodborne illness and hazards. In their journey from farm to factory to fork,
food products encounter many health hazards; and during this journey, the safe food handling
practices are to be implemented and practiced at every stage to prevent risks and harms to the
consumers of such food products.
In India, the Food Safety and Standards Authority of India (FSSAI) has been established
under Food Safety and Standards, 2006, which is a consolidating statute related to food safety
and regulation. FSSAI has been created for laying down scientific standards for articles of
food and to regulate their manufacture, storage, distribution, sale and import to ensure
availability of safe and wholesome food for human consumption.2

8.4.1 Food Safety Guidelines

The World Health Organization has published five keys to safer food, which are as under
i. Keep clean— Washing hands before preparation and during handling of food items is
a must. Sanitizing and protecting the kitchen surface from insects and pests is another
important measure because dangerous microorganisms are widely found in soil, water,
animals and people. These microorganisms are carried on hands, wiping cloths and utensils,
especially cutting boards, and the slightest contact can transfer them to food and cause
foodborne diseases.

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ii. Separate raw & cooked—Raw meat, poultry and seafood must be kept separate from
other foods. Separate utensils, knives etc. should be used for handling such raw foods and
separate containers must be used to separate them from other foods while in storage. Raw
meat, poultry and seafood and their juices may contain harmful micro-organisms which may
be transferred to other foods while in storage and transportation.

iii. Cook thoroughly—foods—especially meat, poultry, eggs and seafood must be

cooked thoroughly as proper cooking kills almost all dangerous micro-organisms. Foods like
soups etc. should be boiled at 70oC at least for one minute.

iv. Keep food at safe temperatures—cooked food must not be left at room temperature
for more than 2 hours as micro-organisms can multiply very quickly if food is so stored. The
cooked food should be refrigerated, preferably below 5oC and should not be stored for long
even in the refrigerator. The food should be kept piping hot (more than 60oC) before serving.

v. Use safe water and raw materials—Raw materials, including water and ice may be
contaminated with dangerous micro-organisms and chemicals, hence safe water must be used
for washing raw material and cooking food. If safe water is not available, it must be made
safe by chlorination, physical filtration. Raw materials can be made safe by washing, peeling
etc.

8.4.2 Food Safety Regulations in India

As mentioned earlier, Food Safety and Standards Authority of India (FSSAI) has been
established under the Food Safety and Standards Act, 2006 which consolidates various Acts
and Orders that have hitherto handled food related issues in various Ministries and
Departments.2
The primary tasks that FSSAI performs2 can be summarized as under—
i. Framing of Regulations to lay down the Standards and guidelines in relation to
articles of food and specifying appropriate system of enforcing various standards thus
notified.
ii. Laying down mechanisms and guidelines for accreditation of certification bodies
engaged in certification of food safety management system for food businesses.
iii. Laying down procedure and guidelines for accreditation of laboratories and
notification of the accredited laboratories.
iv. To provide scientific advice and technical support to Central Government and State
Governments in the matters of framing the policy and rules in areas which have a
direct or indirect bearing of food safety and nutrition.
v. Collect and collate data regarding food consumption, incidence and prevalence of
biological risk, contaminants in food, residues of various contaminants in foods
products, identification of emerging risks and introduction of rapid alert systems.
vi. Creating an information network across the country so that the public, consumers,
Panchayats etc receive rapid, reliable and objective information about food safety and
issues of concern.
vii. Provide training programmes for persons who are involved or intend to get involved
in food businesses.

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viii. Contribute to the development of international technical standards for food, sanitary
and phyto-sanitary standards.
ix. Promote general awareness about food safety and food standards.

8.4.3 Food Safety Guidelines for Food Business Operators by FSSAI—

While at home, we can maintain hygiene to prevent food contamination. But for the foods
that we eat out or the food material that comes to us cooked and packaged i.e. the foods that
are supplied by Food Business Operators (FBOs), FSSAI has set up guidelines5 which can be
summed up as under:

8.4.3.1 Location and Surroundings

• Food Establishment shall ideally be located away from environmental pollution and
industrial activities, to avoid contamination.

8.4.3.2 Layout and Design of Food Establishment Premises

• Layout of the food establishment shall be such that food preparation / manufacturing
processes are not amenable to cross-contamination from other pre and post
manufacturing operations like goods receiving, pre-processing (viz. packaging, washing /
portioning of ready-to-eat food etc).
• Surfaces of floors, ceiling, walls, doors etc. must be smooth and non-absorbent so as to
prevent growth of undesirable moulds.
• Windows, doors etc. should have proper screening to avoid infestation of rodents and
insects. Floors should have proper drainage.
• Drains must be designed so as to prevent entry of rodents from them.

8.4.3.3 Equipment and Containers

• Equipment and containers coming in direct contact with food must be non-corrosive
which do not impart toxicity to the food.
• Utensils and containers must be such as to prevent any toxic gases, odours, dust, dirt,
flies and insects.
• Appropriate facilities for cleaning and disinfection of utensils and equipment must be
arranged.
• Containers and equipment used to hold waste material, cleaning chemicals and other
dangerous substances shall be identified and stored separately to prevent malicious or
accidental contamination of food.
• The fittings / equipment coming in contact with food must be kept in good condition.
Chipped enamelled containers should not be used.

8.4.3.4 Facilities
a. Water Supply- Only potable water, with appropriate facilities of its storage and
distribution, should be used as an ingredient in processing and cooking. Water storage tanks
should be cleaned periodically and non-potable water pipes should be clearly distinguished
from those used for potable water.

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b. Cleaning Utensils / Equipments- adequate facilities like hot and cold water supply,
for cleaning and disinfecting utensils and equipments should be in place.

c. Washing of raw materials-every sink for washing raw materials must have hot / cold
water supply and it must not be used for washing utensils and equipments.

d. Ice and Steam- Ice and steam used in direct contact with food shall be prepared from
potable water in a manner so as to prevent contamination.

e. Drainage and Waste Disposal- food waste and other wastes must be removed from
the place where food is being handled or cooked. The disposal of sewage and effluents shall
be in conformity with requirements of Factory / Environment Pollution Control Board.
Drainage system should be such as to eliminate the contamination of food and water supply.

f. Personnel Facilities and Toilets- Separate toilets for males and females, separate
washing areas with hot / cold water supply should be in place. Number of toilets must be
adequate depending upon the number of employees. Display boards mentioning Do’s and
Dont’s must be put up inside the premises of the food processing facility.

g. Air Quality and Ventilation- Ventilation systems should be installed in a way so that
air does not flow from contaminated areas to clean areas.

h. Lighting- In order to maintain hygiene there should be proper lighting and it should
be ensured that food is not contaminated by breakage of electrical fittings.

i. Food Operations and Controls—

(i) Procurement of raw materials- No raw material or ingredient thereof shall
be accepted by an establishment if it is known to contain parasites, undesirable
microorganisms, pesticides, veterinary drugs or toxic items, decomposed or
extraneous substances. Raw materials must be purchased in quantities that correspond
to storage / preservation capacity. All raw materials must be checked and cleaned
physically and thoroughly. Receiving temperature of potentially high risk food should
be 5ºC or below and that of frozen food should be -18ºC or below.
(ii) Storage of Raw Material and Food- Storage facilities should be designed so
as to protect raw materials and food from contamination, allow maintenance and
cleaning and avoid pest access and accumulation. Cold storage facilities should be
provided wherever needed and raw food especially meat, poultry and seafood shall be
cold stored separately.
Storage of raw materials, ingredients, work-in-progress and processed / cooked or
packaged food products shall be subject to FIFO (First in, First Out), FEFO (First
Expire First Out) stock rotation system as applicable.
Containers made of non-toxic material should be used for storage of raw materials,
processed food etc.

(iii) Food Processing / Preparation, Packaging and Distribution / Service- The

Food Business shall develop and maintain the systems such as time and temperature

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of receiving, processing, cooking, cooling, storage, packaging, distribution and food

service upto the consumer, to ensure the safety and suitability of food.
Whenever frozen food / raw materials are being used / handled / transported, proper
care should be taken so that defrosted / thawed materials are not stored back after
opening for future use.
If thawing is required then only the required portion of food should be thawed at a
time.

(iv) Food Packaging- Packaging materials shall provide protection for all food
products to prevent contamination, damage and shall accommodate required labelling
as laid down under the FSS Act & the Regulations there under. Where food comes in
direct contact with the packaging material, only food grade packaging material should
be used where food comes in direct contact with the packaging material.
(v) Food Distribution / Service- Processed / packaged and ready-to-eat food
shall be protected as per the required storage conditions during transportation and
service. Temperatures and humidity which are necessary for sustaining food safety
and quality shall be maintained. The conveyances and containers shall be designed,
constructed and maintained in such manner that they can effectively maintain the
requisite temperature, humidity, atmosphere and other conditions necessary to protect
food. Containers used for transporting / serving foodstuffs shall be non-toxic, kept
clean and maintained in good condition in order to protect foodstuffs from any
contamination.
Where the same conveyance or container is used for transportation of different foods,
or high-risk foods such as fish, meat, poultry, eggs etc., effective cleaning and
disinfections shall be carried out between loads to avoid the risk of cross-
contamination.

j. Management and Supervision— A detailed Standard Operating Procedure (SOP)

for the processing of food as well as its packing, despatch and storage will be developed for
proper management which in turn would help in identifying any problem and the exact point,
so that damage control would be faster.
The Food Business shall ensure that technical managers and supervisors have appropriate
qualifications, knowledge and skills on food hygiene principles and practices to be able to
ensure food safety and quality of its products, judge food hazards, take appropriate preventive
and corrective action, and to ensure effective monitoring and supervision.

k. Food Testing Facilities— A well-equipped laboratory for testing of food materials /

food for physical, microbiological and chemical analysis in accordance with the
specification/standards laid down under the rules and regulations shall be in place inside the
premises for regular / periodic testing and whenever required.
In case of any suspicion or possible contamination, food materials / food shall be tested
before dispatch from the factory. If there is no in-house laboratory facility, then regular
testing shall be done through an accredited lab notified by FSSAI.
In case of complaints received and if so required, the company shall voluntarily do the testing
either in the in-house laboratory or an accredited lab or lab notified by FSSAI.

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l. Audit, Documentation and Records— Appropriate records of food processing /

preparation, production / cooking, storage, distribution, service, food quality, laboratory test
results, cleaning and sanitation, pest control and product recall shall be kept and retained for a
period of one year or the shelf-life of the product, whichever is more. A periodic Audit of the
whole system shall be done.
m. Sanitation and Maintenance of Establishment Premises—
(i) Cleaning & Maintenance - A cleaning and sanitation programme shall be in
place and the record thereof shall be properly maintained, which shall indicate specific
areas to be cleaned, cleaning frequency and cleaning procedure to be followed,
including equipment and materials to be used for cleaning. Equipment used in
manufacturing will be cleaned and sterilized at set frequencies.
Cleaning chemicals shall be handled and used carefully in accordance with the
instructions of the manufacturer and shall be stored separately away from food
materials, in clearly identified containers, to avoid any risk of contaminating food.

(ii) Pest Control System- Food establishment, including equipment and building
shall be kept in good repair to prevent pest access and to eliminate potential breeding
sites. Holes, drains and other places where pests are likely to gain access shall be kept
in sealed condition or fitted with mesh / grills / claddings or any other suitable means as
required and animals, birds and pets shall not be allowed to enter into the food
establishment areas/ premises.
Food materials shall be stored in pest-proof containers stacked above the ground and
away from walls. Pest infestations shall be dealt with immediately and without
adversely affecting the food safety or suitability. Treatment with permissible chemical,
physical or biological agents, within the appropriate limits, shall be carried out without
posing a threat to the safety or suitability of food. Records of pesticides / insecticides
used along with dates and frequency shall be maintained.

n. Personal Hygiene-
(i) Health Status-Personnel known, or believed, to be suffering from, or to be a
carrier of a disease or illness likely to be transmitted through food, shall not be allowed
to enter into any food handling area . The Food Business shall develop a system
whereby any person so affected, shall immediately report illness or symptoms of illness
to the management and medical examination of a food handler shall be carried out apart
from the periodic checkups, if clinically or epidemiologically indicated.
Arrangements shall be made to get the food handlers / employees of the establishment
medically examined once in a year to ensure that they are free from any infectious,
contagious and other communicable diseases. A record of these examinations signed by
a registered medical practitioner shall be maintained for inspection purposes.
The factory staff shall be compulsorily inoculated against the enteric group of diseases
as per recommended schedule of the vaccine and a record shall be kept for inspection.
In case of an epidemic, all workers are to be vaccinated irrespective of the scheduled
vaccination.

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(ii) Personal Cleanliness- Food handlers shall maintain a high degree of personal
cleanliness. All food handlers should be provided with adequate and suitable clean
protective clothing, head covering, face masks, gloves and footwear and the food
business shall ensure that the food handlers at work should wear only clean clothing
and gear.
Food handlers shall always wash their hands with soap and clean potable water,
disinfect their hands and then dry with hand drier or clean cloth towel or disposable
paper at the beginning of food handling activities immediately after handling raw food
or any contaminated material, tools, equipment or work surface, where this could result
in contamination of other food items or after using the toilet.
Food handlers engaged in food handling activities shall refrain from smoking, spitting,
chewing, sneezing or coughing over any food whether protected or unprotected and
eating in food preparation and food service areas.
The food handlers should trim their nails and hair periodically, do not encourage or
practice unhygienic habits while handling food.
Persons working directly with and handling raw materials or food products shall
maintain high standards of personal cleanliness at all times. In particular, they shall not
smoke, spit, eat or drink in areas or rooms where raw materials and food products are
handled or stored; wash their hands at least each time work is resumed and whenever
contamination of their hands has occurred; e.g. after coughing / sneezing, visiting toilet,
using telephone, smoking etc.; avoid certain hand habits - e.g. scratching nose, running
finger through hair, rubbing eyes, ears and mouth, scratching beard, scratching parts of
bodies etc.- that are potentially hazardous when associated with handling food products,
and might lead to food contamination through the transfer of bacteria from the
employee to product during its preparation. When unavoidable, hands should be
effectively washed before resuming work after such actions.

o. Visitors- Generally visitors should be discouraged from going inside the food
handling areas. The Food Business shall ensure that visitors to its food manufacturing area
must wear protective clothing, footwear and adhere to the other personal hygiene provisions.
p. Product Information and Consumer Awareness— All packaged food products
shall carry a label and requisite information as per provisions of Food Safety and Standards
Act, 2006 and Regulations made there under so as to ensure that adequate and accessible
information is available to each person in the food chain to enable them to handle, store,
process, prepare and display the food products safely and correctly and that the lot or batch
can be easily traced and recalled if necessary.
q. Training— The Food Business shall ensure that all food handlers are aware of their
role and responsibility in protecting food from contamination or deterioration. Food handlers
shall have the necessary knowledge and skills which are relevant to food processing /
manufacturing, packing, storing and serving so as to ensure the food safety and food quality.
The Food Business shall ensure that all the food handlers are instructed and trained in food
hygiene and food safety aspects along with personal hygiene requirements.

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Periodic assessments of the effectiveness of training, awareness of safety requirements and

competency level shall be made, as well as routine supervision and checks to ensure that food
hygiene and food safety procedures are being carried out effectively.
Training programmes shall be routinely reviewed and updated wherever necessary.

8.4 PATENTS
Patent is a special right to the inventor that has been granted by the Government through
legislation for trading new articles. It is a personal property which can be licenced or sold by
the person / organisation just like any other property. The patents give the inventor the rights
to inhibit others from making, using or selling his invention. Hence, a patent is the right
granted by the State to an inventor to exclude others from commercially exploiting the
invention for a limited period, in return for the disclosure of the invention, so that others may
gain the benefit of the invention. The disclosure of the invention is thus an important
consideration in any patent granting procedure.6
The procedure for granting patents, extent of the exclusive rights vary widely between
countries according to national laws and international agreements. The TRIPS (Trade-Related
Aspects of Intellectual Property Rights) Agreement under the World Trade Organization
(WTO) requires Member countries to make patents available for any inventions, whether
products or processes, in all fields of technology without discrimination, subject to the
normal tests of novelty, inventiveness and industrial applicability. It is also required that
patents be available and patent rights enjoyable without discrimination as to the place of
invention and whether products are imported or locally produced (Article 27.1).7

There are three permissible exceptions to the basic rule of patentability:

First - the inventions contrary to ordre public or morality which explicitly includes
inventions dangerous to the human, animal or plant life or health (Article 27.2).7
Second exception is that Members may exclude from patentability diagnostics,
therapeutic and surgical methods for the treatment of humans or animals (Article 27.3(a)).7
Third is that Members may exclude plants and animals other than micro-organisms
and essentially biological processes for the production of plants or animals other than non-
biological processes. However, any country excluding plant varieties from patent protection
must provide an effective sui generis (unique) system of protection (Article 27.3(b)).7
In India, the Indian Patent Act (1970) allows the ‘process patents’ but not the ‘product
patent’, and the maximum duration of patent is for 5 years from the date of grant, and 7 years
from the date of filing the patent application.8

A patent consists of three parts- the grant, specifications and claims.

i. Grant is a signed document and is an agreement that grants patent right to the
inventor. It is filled at the patent office which is not published.
ii. Specification is a narrative in which the subject matter of invention is described as
to how the invention was carried out.
iii. Claim specifically defines the scope of the invention to be protected by the patent
to which the others may not practice.

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The Specification and Claims sections are published as a single document which is
made public at a minimum charge from the patent office.

8.5 TRADE SECRETS

Normally, any confidential business information which gives a competitive edge to an
enterprise may be protected as a trade secret.9 In other words, the private proprietary
information that benefits the owners is called a trade secret.10 Trade secrets are intellectual
property (IP) rights on confidential information which may be sold or licensed as defined by
the World Intellectual Property Organization (WIPO) of the United Nations.
In order to qualify as a trade secret, the information must be commercially valuable, known
only to a limited group of persons and the rightful holder of the information must take
reasonable steps to keep it secret, including the use of confidentiality agreements for business
partners and employees.9 The unauthorized acquisition, use or disclosure of such secret
information is considered dishonest commercial practice, hence it is regarded as a violation of
the trade secret protection.

8.5.1 Types of Information protected by Trade Secrets-

Trade Secrets can be of two types-
● Technical information- such as related to manufacturing processes, pharmaceutical
test data etc.9
● Commercial information- such as distribution methods, list of suppliers and clients
9
etc.
Financial information, recipes, formulas, source codes etc. are other examples of information
that may be protected by trade secrets.9 One of the most popular examples of trade secrets is
Coca Cola that has kept the formulae of its products a secret, hence it has a competitive edge
over other companies.

8.5.2 Trade Secrets in India

There is no specific legislation in India to protect trade secrets. However, at times Indian
Courts have upheld trade secret protection on the basis of principles of equity, breach of
confidence etc. which comes under the Indian Contracts Act, 1872.

8.6 COPYRIGHT
Copyright is a legal term used to describe the rights that creators have over their literary and
artistic works such as books, music, paintings, computer programs, databases, maps,
technical drawings etc.13 Copyright protection extends only to expressions, and not to ideas,
procedures, methods of operation or mathematical concepts as such.13 The material under
copyright cannot be reprinted or reproduced without written permission of the copyright
holders. While patents and trade secrets provide protection of only basic knowhow,
copyrights protect the expressed materials viz., materials in printed, video-recorded or taped
forms.

8.6.1 Copyrights in India-

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The Copyright Act, 1957 has been amended five times since then, i.e., in 1983, 1984, 1992,
1994, 1999 and 2012. The main reasons for amendments to the Copyright Act, 1957 include
to bring the Act in conformity with two WIPO internet treaties concluded in 1996 namely, the
WIPO Copyright Treaty (“WCT”) and WIPO Performances and Phonograms Treaty
(“WPPT”). 14
Section 9 of the Copyright Act requires for establishment of an office to be called the
Copyright Office for the purpose of the Act.14

8.7 TRADEMARKS
A trademark is an identification symbol which is used in the course of trade to enable the
public to distinguish one trader's goods from the similar goods of other traders. The public
makes use of the trademarks to choose from a plethora of products and then repeat their
orders by using the trademarks.15

8.7.1 Trade-Mark Registry in India-

The Trade Marks Registry was established in India in 1940 and presently it
administers the Trade Marks Act, 1999 and the rules made thereunder. It acts as a resource
and information Centre and is a facilitator in matters relating to trademarks in the country.16

8.8 ETHICAL ISSUES

Ethics is the discipline concerned with what is good or bad, right or wrong.17 The widespread
development of Biotechnology, life sciences, genetic engineering and exploitation of genetic
resources have brought the focus on the ethical angle of these techniques. The misuse of
Biotechnology and related technologies may result in loss of human life, threat to human
health, environmental hazards, political & financial losses etc. to institutions and
Governments. To avoid and minimize such harms, the ethical dimension of life sciences
needs to be taken care of. Beside others, the most talked about aspect is the ethical
implication of protecting biotechnological inventions through the Intellectual Property
System.

Some general principles involved in ethical concerns in context of protecting Intellectual

Property are17-
● transparency and access to information.
● prior informed consent.
● access to fruits of technology.
● pluralism, or accommodation of different value systems.
There may arise some ethical questions, examples of which may be such as - Should research
on human cloning be permitted? Should patents be granted for DNA sequences? Should
genetically modified organisms (GMOs) be allowed?, and so on.

8.9 PLANT GENETIC RESOURCES

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According to the National Bureau of Plant Genetic Resources (NBPGR), Plant Genetic
Resources (PGR) are any living material of present and potential value for humans. PGR
includes all our agricultural crops and some of their wild relatives because they possess some
valuable traits. Occasionally, genes, DNA fragments and RNA are also included under the
purview of Genetic Resources.

8.9.1 Conservation of Plant Genetic Resources-

According to the Food & Agricultural Organization (FAO) of the United Nations, plant
diversity is being eroded due to use of modern varieties and reduction in the number of
cultivars. Modern agriculture, which is intensive and focussed on increased production, has
resulted in a narrow genetic base for the crops grown. While, traditional agriculture had a
large number of diverse crop varieties. Plant Genetic Resources provide valuable traits
needed to overcome the dangers of extinction of important plant species, being the only
source of plant genetic diversity.
So, to conserve the diversity found within species of cultivated plants, experts employ a
strategy that combines ex-situ conservation (storing diversity in genebanks) with in-situ on-
farm conservation.
Examples-
The Thrissur centre of NBPGR in Kerala is responsible for collection and evaluation of
germplasm of southern peninsular region with particular emphasis on spices and plantation
crops.

8.10 PLANT VARIETAL PROTECTION AND REGISTRATION

As per World Intellectual Property Organization, Plant variety protection, also called a “plant
breeder’s right,” is a form of intellectual property right granted to the breeder of a new plant
variety. The International Union for the Protection of New Varieties of Plants, known as
“UPOV” was established by the International Convention for the Protection of New Varieties
of Plants. It was adopted in Paris in 1961. In that way, the intellectual property rights of plant
breeders were recognized for their crop varieties on an international basis. In order to
accelerate agricultural development, it is necessary to protect the rights of farmers and plant
breeders, who conserve & improve old varieties and develop new varieties of important crop
plants.

8.11 GATT and TRIPS

8.11.1 GATT
GATT stands for General Agreement on Tariffs & Trade and in a way it promotes trade
liberalization. For the first time it was agreed upon in 1948 between 23 countries. It remained
in effect till 1995 when its members were 128 countries. It was replaced by the World Trade
Organization (WTO). It was framed by the developed countries to get rid of conflicts that
arise among the countries for getting a share in world trade. However, it is considered that,
for a long time, the benefits from GATT were achieved only by the developed countries.

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The GATT agreement functions through the Goods Council (Council for Trade in Goods)
which comprises representatives from all WTO members.18 GATT covers international trade
of goods through multilateral agreements between the member countries. The primary
purpose of GATT was to promote international trade by reducing or removing trade barriers
such as tariffs and practices of trade protectionism.

8.11.2 TRIPS
TRIPS (Trade-Related Aspects of Intellectual Property Rights) Agreement was negotiated
during the Uruguay Multilateral Trade Negotiations (MTN) Round, which introduced the
intellectual property rules into the multilateral trading system for the first time. It is
administered by the WTO and considered the most comprehensive multilateral agreement on
intellectual property (IP). As mentioned earlier, TRIPS agreement facilitates the availability
of patents for inventions, whether products or processes, in all fields of technology without
discrimination.

8.12 PATENTING AND BIOLOGICAL MATERIAL

Like other inventions, an invention in the field of biology can also be patented as per
prevalent law. The processing of patents of biological materials varies across different
jurisdictions (of different countries). Generally, biological inventions are patented like
chemical ones. These include biological processes like processes for producing useful
products like proteins, enzymes etc., products like genetically modified organisms (GMOs),
composition or formulations, methods etc.
Patenting of biological material and liveforms was earlier not permitted, however, the laws
were amended later on. For example- Oncomouse, which is a genetically engineered mouse,
containing human cancer mouse was developed in the United States. Patent was issued in
favour of this liveform and cleared the way for future patents for inventions in biology. In the
wake of this patent, a superbug was allowed patent, which was developed by Dr. Anand
Mohan Chakrabarty, an Indian born American scientist. Dr. Chakrabarty was allowed to treat
oil spills by using the superbug that was developed by using a bacterium, Pseudomonas that
was capable of degrading hydrocarbons (present in the oil). This superbug was developed by
inserting into the bacteria multiple circular DNA molecules (known as plasmids), each with
genes encoding different enzymatic functions in hydrocarbon degradation, he and his team
were able to create a new variety of Pseudomonas that could degrade crude oil in Petri
dishes.19
Other examples of patents in biology are 20, 21
i. Genetically engineered E. coli in which human genes for insulin, growth hormone etc. were
introduced.
ii. Recombinant Mouse-Human Chimeric fab Against Hepatitis B Surface Antigen.
iii. Recombinant Mouse-Human Chimeric fab Against Hepatitis B Surface Antigen.
iv. Bollworm-resistant cotton, insect resistant tobacco etc.

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8.13 BIO-SAFETY AND CONTAINMENT PRACTICES

Modern biotechnology has evolved for the benefit of humankind. If handled properly,
biotechnology can be the most important technological tool to eradicate all the problems
related to human health, environment, food production etc. But if the protocols of biosafety
are not followed and policies and procedures are not set in place, the boon of biotechnology
may turn into the most destructive and harmful inventions of man. We have recently seen this
in the form of Covid-19 pandemic, where it is believed that the Corona virus has spread from
a laboratory in Wuhan, China.
Biosafety includes a range of measures, policies and procedures so as to minimize potential
hazards that Biotechnology may pose to the Human health and environment. The Cartagena
Protocol on biosafety to the Convention on Biological Diversity (CBD) is an international
agreement which aims to ensure the safe handling, transport and use of living modified
organisms (LMOs) resulting from modern biotechnology that may have adverse effects on
biodiversity, taking also into account risks to human health.22
The protocol primarily deals with GMOs that are to be introduced into the environment (such
as seeds, trees or fish) and does not cover pharmaceuticals for humans addressed by other
international agreements and organizations.23

8.14 FOOD SAFETY OF GM CROPS

From the discussion done so far, you are now aware that the foods that have been derived
from organisms whose genetic material (DNA) has been modified artificially are Genetically
Modified (GM) foods e.g. genetically engineered papaya is ‘ring spot virus resistant’, thus
with high productivity. And, the purpose of GM crops has been to increase productivity and
the major concern has been to mitigate the health-related worries involved. Despite many
concerns, we already have many drugs, foods and other consumables that have been derived
from genetic modification of organisms. Also, before any food product is launched into the
market, it undergoes rigorous tests and animal feeding trials. You are now aware that there is
a set of regulations and legislations put in place by our governments and authorities which
allow only the tested and approved GM foods on our plates.
An example regarding GM foods is well known and worth a quote here.
• In 1998, a Scottish scientist Dr. Pusztai published his research findings based on his lab
experiments with GM potatoes and rats. His experiments were to find out whether the
protein, known as lectin-found in plants, was harmful to rats. Lectin was investigated so as
to introduce pest resistance in crops.
• His findings involved three groups of rats. One group ate non-GM crops laced with
lectins, another group ate the potatoes that were genetically modified to produce their own
lectins and the third group ate non-GM i.e. conventional potatoes.
• Based on his experiments, Dr. Pusztai claimed that the rats eating GM potatoes had
smaller livers and brains, larger spleens and had suppressed immune systems. And, the
rats that ate non-GM potatoes showed no such side-effects.

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• These conclusions of Dr. Pusztai were, however, declined by a group of four independent
bodies including the Royal Society.
•
The Royal Society, London claims that, so far there is no evidence that a crop is dangerous
because it is GM and since widespread commercialization of GM products, there has been
no evidence of ill effects of consumption of any approved GM crop.24
•
Moreover, an animal feeding trial of GM tomatoes modified to produce high levels of
antioxidants showed the GM tomatoes reduced the levels of cancer. This is not because the
tomatoes are GM, but rather because they produce antioxidants, which are known to
reduce cancer.24

8.15 SUMMARY
Food is the primary need for sustenance of a healthy life of all the living organisms. Today’s
world order is rapid development and the major obstacle in poverty and ever-increasing
demand of food grain. The development of life sciences and particularly biotechnology has
helped us to satiate the hunger of millions to a large extent. Today we have high yield, pest
resistant and nutrient rich superfoods just because of genetic engineering. Many cross bred
vegetable crops have added to our delicacies. The bio-safety and food-safety guidelines of
nations are set in place which regulates the introduction of GM foods in our food chain.
Intellectual Property Rights including patents, copyrights, trademarks etc. are there to lessen
the trade related arguments and to mitigate the losses to the worthy. GATT (now WTO) and
TRIPS have provided trade liberalization by providing equal share in trade of products. For
the conservation of genetic diversity, the National Bureau of Plant Genetic Resources
(NBPGRs) have been set up all over India, which are responsible for the in-situ and ex-situ
conservation and preservation of genetic resources.

8.16 GLOSSARY
1. Bt - Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, the
most commonly used biological pesticide worldwide. Bt Brinjal is an example where gene
from this bacterium has been inserted.
2. Transgenic organisms - organisms that have been developed or modified by the use
or Recombinant DNA technology.
3. Virulence - the severity or harmfulness of a disease or poison.
4. Pathogenesis - the manner of development of a disease.
5. Sui generis- constituting a class alone:Unique, Peculiar
6. Ordre public- The term ‘ordre public’, derived from French law, expresses concerns
about matters threatening the social structures which tie a society together, i.e., matters that
threaten the structure of civil society as such.
7. in-situ conservation-conservation of species in their natural habitat.
8. ex-situ conservation-An approach to the conservation of biodiversity that is based on
keeping organisms and species alive by the deliberate removal of biological resources (seed,
pollen, sperm, individual organisms) from their original habitat or natural environment, and
protecting them elsewhere under controlled conditions.

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8.17 SELF ASSESSMENT QUESTIONS

8.17.1 Objective Type

1. In the phrase ‘GM crops’, GM stands for—
(i) Geographically Modified (ii) Geologically Modified
(iii) Genetically Modified (iv) None of the above.
2. In GM cropssuch as BtCotton, Bt Brinjal etc., Btstands for—
(i) Bacterium - Bacillus thuringiensis (ii) Virus - Bacillus thuringiensis
(iii) Fungus - Bacillus thuringiensis (iv) All of the above.
3. With regard to consumption of food produce from GM crops, our scientists are mainly
concerned about—
(i) Bio- and Food-safety of food (ii) Market value of food produce,
(iii) Nutritional value of food (iv) Scientists are not at all concerned.
4. In India, which Ministry / agency had laid down rules and procedures,in 1989, for the
manufacture, import, use and release of GMOs and the products obtained from such
organisms—
(i) Ministry of Environment, Forest and Climate Change (MoEF& CC)
(ii) Recombinant DNA Advisory Committee (RDAC)
(iii) Department of Biotechnology (DBT).
(iv) Ministry of Law & Justice.
5. Which Government Agency has been created for laying down scientific standards for
articles of food and to regulate their manufacture, storage, distribution, sale and import to
ensure availability of safe and wholesome food for human consumption, in India—
(i) Review Committee on Genetic Manipulation (RCGM)
(ii) Genetic Engineering Appraisal Committee (GEAC)
(iii) Recombinant DNA Advisory Committee (RDAC)
(iv) Food Safety and Standards Authority of India (FSSAI)
6. Which international agreement requires Member countries to make patents available for
any inventions, whether products or processes, in all fields of technology without
discrimination, subject to the normal tests of novelty, inventiveness and industrial
applicability—
(i) General Agreement on Tariffs and Trade (GATT)
(ii) Agreement on Trade-Related Aspects of Intellectual Property Rights
(iii) Trade-Related Aspects of Intellectual Property Rights (TRIPS)
(iv) All of the above.
7. A patent consists of which three parts—
(i) Grant, specifications and claims (ii) Cost, speciality and effects
(iii) Subject, material and awareness (iv) None of the above.
8. With regard to Trademark, choose the appropriate option–
(i) It is an identification symbol which is used in the course of trade to enable the
public to distinguish one trader's goods from the similar goods of other traders.

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(ii) The public makes use of the trademarks to choose from a plethora of products
and then repeat their orders by using the trademarks.
(iii) Both of the above are correct.
(iv) None of the above is correct.
9. Any confidential business information which gives a competitive edge to an enterprise is
known as –
(i) Patent (ii) Trade Secret
(ii) Intellectual Property (IP) (iv) Copyright
10. By inserting a gene from the soil Bacterium Bacillus thuringiensis into the genome of
various brinjal cultivars,BtBrinjal was developed by—
(i) Ministry of Environment, Forest and Climate Change (MoEF& CC)
(ii) Maharashtra Hybrid Seeds Company (Mahyco)
(iii) National Bureau of Plant Genetic Resources (NBPGR)
(iv) Govind Ballabh Pant University of Agriculture & Technology, Pantnagar.
8.17.2 Subjective Type
1. Why do we need Genetically Modified organisms and crops?
2. What are the concerns of some scientists about the use of genetically engineered
organisms?
3. Summarize the recommendations of Recombinant Advisory Committee (RAC) established
by the National Institutes of Health (NIH), USA.
4. What is Bio-safety. Elaborate the objectives of Biosafety guidelines?
5. How the Bio-safety guidelines are being implemented in India?
6. What do you mean by Food-safety?
7. Discuss the Food-safety guidelines as issued by World Health Organization (WHO).
8. Describe the functions and role of Food Safety and Standards Authority of India (FSSAI).
9. What are patents. Explain different parts of patent?
10. Define – (i) Copyright (ii) Trademark (iii) Trade Secrets.
11. What ethical issues are involved in the use of Biotechnology?
12. Why Plant Genetic Resources are important. How Plant Genetic Resources are conserved
through National Bureau of Plant Genetic Resources (NBPGR).
13. What is the significance of GATT and TRIPS in promoting trade among nations?
14. Describe the food-safety concerns related to use of GM crops?

8.18 REFERENCES
1 WHO, Laboratory Biosafety Manual (3rd ed.) (2004), available at
http://www.who.int/ihr/publications/WHO_CDS_CSR_LYO_2004_11/en/index.html,
p.47
2 https://fssai.gov.in/cms/about-fssai.php
3 https://www.who.int/health-topics/food-safety/
4 https://www.who.int/foodsafety/publications/consumer/manual_keys.pdf
5 Lic. regin-020811.pmd (fssai.gov.in)

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6 https://web.archive.org/web/20130520221306/http://www.wipo.int/export/sites
/www/about-ip/en/iprm/pdf/ch2.pdf, pp. 17
7 https://www.wto.org/english/tratop_e/trips_e/intel2_e.html
8 Dubey R.C. (2006). A Textbook of Biotechnology, pp. 606-607, S.Chand& Company
Ltd.
9 https://www.wipo.int/tradesecrets/en/
10 Dubey R.C. (2006). A Textbook of Biotechnology, pp. 608, S.Chand& Company Ltd.
11 https://ipindia.gov.in/writereaddata/Portal/IPOAct/1_31_1_patent-act-1970-11march
2015.pdf
12 Dubey R.C. (2006). A Textbook of Biotechnology, pp. 607-608, S.Chand& Company
Ltd.
13 https://www.wipo.int/copyright/en/
14 https://copyright.gov.in/
15 Dubey R.C. (2006). A Textbook of Biotechnology, pp. 608, S.Chand& Company Ltd.
16 https://ipindia.gov.in/about-us-tm.htm
17 https://www.wipo.int/edocs/pubdocs/en/intproperty/932/wipo_pub_b932ipb.pdf
18 https://www.wto.org/english/tratop_e/gatt_e/gatt_e.htm
19 https://www.natureasia.com/en/nindia/article/10.1038/nindia.2020.192
20 https://dbtindia.gov.in/patent-granted-years-2012-2013-2014-2015
21 Dubey R.C. (2006). A Textbook of Biotechnology, pp. 613, S.Chand& Company Ltd.
22 The Cartagena Protocol on Biosafety under the Convention on Biological diversity -
https://bch.cbd.int/protocol/
23 United Nations Environment Programme. Biosafety and precaution, pp.5,
https://www.cbd.int/doc/press/presskits/bs/cpbs-unep-cbd- en.pdf
24 https://royalsociety.org/topics-policy/projects/gm-plants/is-it-safe-to-eat-gm-crops/

8.19 SUGGESTED READINGS

1. Food Safety Management Programs: Applications, Best Practices, and Compliance by
Debby Newslow.
2. "Biosafety and biosecurity memo" (PDF). whitehouse.gov.
3. Intellectual Property Law by P Narayanan.
4. Introduction to Intellectual Property Rights by H.S. Chawla.
5. Plant Genetic Resources in Indian Perspective - Theory and Practices by B P
Singh and Umesh Srivastava.

8.20 TERMINAL QUESTIONS

1. Is it possible in practice to avoid the bio-hazards and food-hazards in the present
polluted environment?
2. Will more pandemics, like Covid-19, hit the human race in future?
3. How to implement the principles of Bio-ethics effectively so as to reduce the hazards
to human health and environment?

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8.21- ANSWERS TO OBJECTIVE-TYPE QUESTIONS

1. (iii) Genetically Modified.
2. (i) Bacterium - Bacillus thuringiensis.
3. (i) Bio- and Food-safety of food.
4. (i) Ministry of Environment, Forest and Climate Change (MoEF& CC).
5. (iv) Food Safety and Standards Authority of India (FSSAI).
6. (iii) Trade-Related Aspects of Intellectual Property Rights (TRIPS).
7. (i) Grant, specifications and claims.
8. (iii) Both of the above are correct.
9. (ii) Trade Secret.
10. (ii) Maharashtra Hybrid Seeds Company (Mahyco).

Abbreviations—
1. GE – Genetically Engineered.
2. GM- Genetically Modified
3. GMOs - Genetically Modified Organisms.
4. Mahyco - Maharashtra Hybrid Seeds Company.
5. FSB - Fruit & Shoot Borer pest.
6. NIH - National Institutes of Health, USA.
7. RAC - Recombinant Advisory Committee.
8. UNEP - United Nations Environment Programme.
9. WHO – World Health Organization.
10. MoEF& CC - Ministry of Environment, Forest and Climate Change.
11. RDAC - Recombinant DNA Advisory Committee.
12. EPA - Environment Protection Act.
13. IBC - Institutional Biosafety Committee.
14. RCGM - Review Committee on Genetic Manipulation.
15. DBT – Department of Biotechnology.
16. SBCC - State Biotechnology Coordination Committee.
17. DLC - District Level Committee.
18. GEAC - Genetic Engineering Appraisal Committee.
19. FSSAI - Food Safety and Standards Authority of India.
20. FBOs - Food Business Operators.
21. FIFO - First in, First Out.
22. FEFO - First Expire First Out.
23. SOP - Standard Operating Procedure.
24. TRIPS - Trade-Related Aspects of Intellectual Property Rights.
25. WTO - World Trade Organization.
26. IPR - Intellectual Property Rights.
27. WIPO - World Intellectual Property Organization.
28. WPPT - WIPO Performances and Phonograms Treaty.
29. WCT - WIPO Copyright Treaty.
30. NBPGR - National Bureau of Plant Genetic Resources.

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31. PGR - Plant Genetic Resources.

32. FAO - Food & Agricultural Organization.
33. UPOV - International Union for the Protection of New Varieties of Plants.
34. GATT - General Agreement on Tariffs & Trade.
35. CBD - Convention on Biological Diversity.

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UNIT-9 BIOTECHNOLOGICAL APPLICATIONS

Contents
9.1 Objectives
9.2 Introduction
9.3 Genetic engineering
9.4 Prevention of Genetic Disorders
9.5 Treatment of Diseases and Genetic Disorders
9.6 Biotechnological applications in agriculture
9.6.1 Biofertilizers
9.6.2. N2 fixers
9.6.3 Phosphate solubilizers
9.6.4. Phosphate Mobilizer
9.6.5 Importance of biofertilizers
9.6.6 Rhizobium
9.6.7 Biotechnological applications for horticulture
9.6.8 Biotechnological applications for Forestry
9.6.9. Biotechnological applications for medicines
9.6.9.1 Recombinant Insulin
9.6.9.2 Gene Therapy
9.6.9.3 Molecular Diagnosis
9.6.9.4 Pharmacogenomics
9.6.9.5 Edible Vaccines
9.7 Biotechnological applications for industry
9.8 Fermentation
9.8.1 Definitions of Fermentation
9.8.2 Examples of Fermentation
9.9 Summary
9.10 Bibliography
9.11 Terminal Questions

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9.1 OBJECTIVES
The introduction of the unit will discuss biotechnological applications. The unit describes the
detailed applications of biotechnology in agriculture, horticulture, forestry, medicines, and
industries. The unit will be beneficial for the teaching and, learning of undergraduate and
postgraduate courses.

9.2 INTRODUCTION
Biotechnology refers to the combination of biological sciences and technology. The term
biotechnology was first given by Karoly Erekyin 1919,meaning the production of products
from raw materials with the aid of living organisms. The concept of biotechnology
encompasses a wide range of procedures for modifying living organisms according to human
purposes, going back to the domestication of animals, cultivation of plants, and
"improvements" to these through breeding programs that employ artificial selection and
hybridization. Modern usage also includes genetic engineering as well as cell and animal and
plant tissue culture technologies. The American Chemical Society defines biotechnology as
the application of biological organisms, systems, or processes by various industries to
learning about the science of life and the improvement of the value of materials and
organisms such as pharmaceuticals, crops, and livestock. According to the European
Federation of Biotechnology (EFB), biotechnology is the integration of natural science and
organisms, cells, parts thereof, and molecular analogs for products and services.
Biotechnology is based on the basic biological sciences (e.g., molecular biology,
biochemistry, cell biology, embryology, genetics, microbiology) and conversely provides
methods to support and perform basic research in biology.
Biotechnology is the research and development in the laboratory using bioinformatics for
exploration, extraction, exploitation, and production from any living organisms and any
source of biomass by means of biochemical engineering where high value-added products
could be planned (reproduced by biosynthesis, for example), forecasted, formulated,
developed, manufactured, and marketed for the purpose of sustainable operations (for the
return from bottomless initial investment on R & D) and gaining durable patents rights (for
exclusives rights for sales, and prior to this to receive national and international approval
from the results on animal experiment and human experiment, especially on the
pharmaceutical branch of biotechnology to prevent any undetected side-effects or safety
concerns by using the products).The utilization of biological processes, organisms or systems
to produce products that are anticipated to improve human lives is termed biotechnology.
By contrast, bioengineering is generally thought of as a related field that more heavily
emphasizes higher systems approaches (not necessarily the altering or using of biological
materials directly) for interfacing with and utilizing living things. Bioengineering is the
application of the principles of engineering and natural sciences to tissues, cells, and
molecules. This can be considered as the use of knowledge from working with and
manipulating biology to achieve a result that can improve functions in plants and animals.
Relatedly, biomedical engineering is an overlapping field that often draws upon and applies
biotechnology (by various definitions), especially in certain sub-fields of biomedical or

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chemical engineering such as tissue engineering, biopharmaceutical engineering, and genetic

engineering.

9.3 GENETIC ENGINEERING

Genetic engineering, recombinant DNA technology and biotechnology – the buzz words you may
have heard often on radio or TV, or read about in featured articles in newspapers or popular
magazines. It is a set of techniques that are used to achieve one or more of three goals: to reveal the
complex processes of how genes are inherited and expressed, to provide better understanding and
effective treatment for various diseases, (particularly genetic disorders) and to generate economic
benefits which include improved plants and animals for agriculture, and efficient production of
valuable biopharmaceuticals. The characteristics of genetic engineering possess both vast promise and
potential threat to human kind. It is an understatement to say that genetic engineering will
revolutionize the medicine and agriculture in the 21st future. As this technology unleashes its power
to impact our daily life, it will also bring challenges to our ethical system and religious beliefs.
Genetic Engineering and Human Health Soon after the publication of the short essay by Crick and
Watson on DNA structure (1953), research began to uncover the way by which DNA molecules can
be cut and “spliced” back together. With the discovery of the first restriction endonuclease by
Hamilton Smith et al. (1970), the real story of genetic engineering began to unfold. The creation of
the first engineered DNA molecule through splicing DNA fragments of two unrelated species together
was made public in 1972. Soon followed were a whole array of recombinant DNA molecules,
genetically modified bacteria, viruses, fungi, plants and 2 animals. The debate over the issues of
“tinkering with God” heated up and public outcry over genetic engineering was wide-spread. The
birth of “Dolly”, the first mammal ever cloned from an adult body cell, has elevated the debate over
the impact of biological research to a new level. Furthermore, a number of genetically modified
organisms (GMOs) have been commercially released since 1996. Today, it is estimated that over 70%
of US foods contain some ingredients from GMOs. Obviously, genetic engineering holds tremendous
promise for medicine and human well-being. Medical applications of genetic engineering include
diagnosis for genetic and other diseases; treatment for genetic disorders; regenerative medicine using
pluripotent (stem) cells; production of safer and more effective vaccines, and pharmaceuticals; the
prospect of curing genetic disorders through gene therapy; the list goes on... Owing to its potential to
give humanity unprecedented power over life itself, the research and application of genetic
engineering has generated much debate and controversy. Many human diseases, such as cystic
fibrosis, Downs syndrome, fragile X syndrome, Huntington’s disease, muscular dystrophy, sickle-cell
anemia, Tay-Sachs disease, etc. are inherited. There are usually no conventional treatments for these
disorders because they don’t respond to antibiotics or other conventional drugs. Another area is the
commercial production of vaccines and pharmaceuticals through genetic engineering, which has
emerged as a rapidly developing field. The potential of embryonic stem cells to become any
cell/tissue/organ under adequate conditions holds enormous promise for regenerative medicine.

9.4 PREVENTION OF GENETIC DISORDERS

Although prevention may be achieved by avoiding these environmental factors that cause the
abnormality, the most effective prevention, when possible, is to reduce the frequency of or
eliminate entirely the harmful genes (mutations) from the general population. As more
precise tools and procedures for manipulating individual genes are optimized, this will
eventually become a reality. The prevention of genetic disorders at present is usually
achieved by ascertaining those individuals in the population who are at risk of passing a

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serious genetic disorder to their offspring, offering them genetic counselling and prenatal
screening followed with the selective abortion of affected foetuses. Genetic counselling is the
processes of communicating information gained through classic genetic studies and
contemporary research to those individuals who are themselves at risk or have a high
likelihood of passing defects to their offspring. During counselling, information about the
disease itself - its severity and prognosis, whether or not there are effective therapies, and the
risk of recurrence is generally presented. For those couples who find the risks unacceptably
high, counselling may also include discussions ofcontraceptive methods, adoption, prenatal
diagnosis, possible abortion and artificial insemination by a donor, etc. Even though the final
decision must still rest with the couple themselves, the significant increase in the accuracy of
risk assessment made possible with genetic technology makes it easier for parents to make
well-informed decisions. To these couples who find the burden of having an affected child
unbearable, prenatal diagnosis may solve their dilemma. Prenatal screening could be
performed for a varietyof genetic disorders. It requires samples of foetal cells or chemicals
produced by the foetus through either amniocentesis or chorionic villus sampling. After
sampling, severalanalyses could be performed. First, biochemical analysis is used to
determine theconcentration of chemicals in the sample and therefore diagnose whether a
particular foetus is deficient or low in enzymes that facilitate specific biological reactions.
Next, analysis of the chromosomes of the foetal cells can show if all the chromosomes
arepresent, and whether or not there are any structural abnormalities in any of them. Finally,
the most effective means is to detect the defective genes through recombinant DNA
techniques. This has become possible with the rapid increase of DNA copies through
atechnique called PCR, which can produce virtually unlimited copies of a specific gene
orDNA fragment, starting with as little as a single copy. Routine prenatal diagnosis isbeing
performed to screen foetus for Down syndrome, Huntington’s disease, sickle-cell anaemia
and Tay-Sachs disease. Procedures are being developed for prenatal diagnosis of more and
more severe genetic disorders. Thus, an effective roadblock to the passing ofdefective genes
from one generation to another in the population is possible.

9.5 TREATMENT OF DISEASES AND GENETIC DISORDERS

Genetic engineering may be used for direct treatments of diseases or genetic disorders
through various means, including the production of possible vaccines for AIDS, treatment for
various cancers, synthesis of biopharmaceuticals for a variety of metabolic, growth and
development diseases, etc. In general, biosynthesis is a process where gene coding for a
particular product is isolated, cloned into another organism (mostly bacteria), and later
expressed in that organism (host). By cultivating host organism, large quantities of the gene
products can be harvested and purified. A few examples will illustrate the useful features of
biosynthesis. Insulin is essential for the treatment of insulin-dependent diabetes, the most
severe form of diabetes. Historically, insulin was obtained from a beef or pig pancreas. Two
problems exist for the traditional supply of insulin. First, large quantities of the pancreas are
needed to extract enough insulin for continuous treatment of one patient. Second, insulin so
obtained is not chemically identical to human insulin, hence some patients may produce
antibodies which can seriously interfere with the treatment. Human insulin produced through

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genetic engineering is quite effective yet without any side-effects. It has been produced
commercially and made available to patients since 1982. Another successful story in
biosynthesis is the production of human growth hormone (HGH), which is used in the
treatment of children with growth retardation called pituitary dwarfism. The successful
biosynthesis of HGH is important due to several reasons. The conventional source of HGH
was human pituitary glands removed at autopsy, which only exist in brain and liver. Each
child afflicted with pituitary dwarfism needs twice-a-week injections until the age of 20. Such
a treatment regime requires over a thousand pituitaries. It’s obvious that autopsy supply could
hardly keep up with the demand. Furthermore, due to a small amount of virus contamination
in the extracted HGH, many children receiving treatment developed virus related diseases.
Other biopharmaceuticals under development or in pre-clinical or clinical trials through
genetic engineering include anti-cancer drugs, anti-aging agents and a possible vaccine for
AIDS, malaria, etc. Broadly speaking, three types of gene therapy exist, germ line therapy,
enhancement gene therapy and somatic gene therapy. All gene therapy trials currently
underway or in the pipeline are restricted to the somatic cells as targets for gene transfer. The
germ line therapy involves the introduction of novel genes into germ cells such as egg/early.

9.6 BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE

The agricultural biotechnology sector shares a common scientific foundation with the
therapeutic biotechnology sector, including similar characteristics of a lengthy time to market
for emerging products. But the challenges, goals, and opportunities for agricultural
applications of biotechnology provide a very different context for innovation and
entrepreneurs. Now just 30 years old, the agriculture biotechnology sector is entering its third
cycle of innovation, with the first wave being agricultural biotechnology trait creation
followed by a second wave of agriculture biotechnology trait commercialization. The
agricultural trait segment continues to focus on two product categories, herbicide tolerance
and insect resistance, in spite of few entrepreneurial opportunities available. However,
agriculturebiotechnology applications to chemistries, biopesticides, microbials, and natural
products offer new opportunities. These sectors offer new business opportunities that, like the
first wave of agriculture biotechnology, enable entrepreneurs to think about creating
disruptive businesses, not just disruptive technologies for today’s business models.
Agriculture biotechnology uses the bacterial, fungal and algal cultures for the crop
improvement and yield enhancement. The microbial strains present into the soil and water
environment is called the indigenous cultures due to their natural presence. These indigenous
microbial cultures present in soil as well as in plant parts. In soil they are known as free
living microorganism, whereas in plant root regain called as rhizospheric microbes.
Rhizospheric microbes are specific because they are attracting due to the plant root exudates.
In this way microbes make a special type of interaction with plants is known as plant microbe
interactions. The PGP microbial strains help to enhance the crops yield and agricultural
productivity. Plant rhizosphere provide the mechanical support and facilitating the water and
nutrient uptake.
These compounds secreted by plant roots act as chemical attractants for a vast number of
heterogeneous, diverse and actively metabolizing soil microbial communities. The chemicals

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which are secreted by roots into the soils are generally called as root exudates. The exudation
of a wide range of chemical compounds modifies the chemical and physical properties of the
soil and thus, regulates the structure of soil microbial community in the immediate vicinity of
root surface. In fact, some of the exudates act as repellants against microorganisms while
others act as attractants to lodge the microbes. The composition of these exudates is
dependent upon the physiological status and species of plants and microorganisms.
Moreover, these exudates also promote the plant-beneficial symbiotic interactions and inhibit
the growth of the competing plant species. Also, microbial activity in the rhizosphere affects
rooting patterns and the supply of available nutrients to plants, thereby modifying the quality
and quantity of root exudates. A fraction of these plant-derived small organic molecules is
further metabolized by microorganisms in the vicinity as carbon and nitrogen sources, and
some microbe-oriented molecules are subsequently re-taken up by plants for growth and
development. Indeed, carbon fluxes are critical determinants of rhizosphere function. It is
reported that approximately 5–21% of photosynthetically fixed carbon is transported to the
rhizosphere through root exudation. Thus, the rhizosphere can be defined as any volume of
soil specifically influenced by plant roots and/or in association with roots hairs, and plant-
produced materials. Largely, three separates but interacting components are recognized in the
rhizosphere: the rhizosphere (soil), the rhizoplane, and the root itself. Of these, the
rhizosphere is the zone of soil influenced by roots through the release of substrates that affect
microbial activity. The rhizoplane, on the other hand, is the root surface including the
strongly adhering soil particles while the root itself is a component of the system, because
many micro-organisms (like endophytes) also colonize the root tissues. Microbial
colonization of the rhizoplane and/or root tissues is known as root colonization, whereas the
colonization of the adjacent volume of soil under the influence of the root is known as
rhizosphere colonization.

9.6.1 Biofertilizers
Biofertilizers are defined as preparations containing living cells or latent cells of efficient
strains of microorganisms that help crop plants uptake of nutrients by their interactions in the
rhizosphere when applied through seed or soil. They accelerate certain microbial processes in
the soil which augment the extent of availability of nutrients in a form easily assimilated by
plants. Use of biofertilizers is one of the important components of integrated nutrient
management, as they are cost effective and renewable source of plant nutrients to supplement
the chemical fertilizers for sustainable agriculture. Several microorganisms and their
association with crop plants are being exploited in the production of biofertilizers. They can
be grouped in different ways based on their nature and function.

9.6.2 N2 fixers
(a) Free living: Aerobic- Azotobacter, Beijerinckia, Anabaena; Anaerobic- Clostridium Faultative
anaerobic- Klebsiella
(b) Symbiotic: Rhizobium, Frankia, Anabaena azollae
(c) Associative symbiotic: Azospirillum
(d) Endophytic: Gluconacetobacter, Burkholdria

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9.6.3 Phosphate solubilizers

(a) Bacteria: Bacillus megaterium var. phosphaticum; B. subtilis, B. circulans; Pseudomonas
striata
(b) Fungi: Penicillium spp. Aspergillus awamori

9.6.4 Phosphate Mobilizer

The Arbuscular Mycorrhizae (AM) fungi, Ectomycorrhizal fungi, ericoid mycorrhiza and
orchid mycorrhizae can play an important role in phosphate mobilization.

9.6.5 Importance of biofertilizers

Biofertilizers are known to make a number of positive contributions in agriculture.
• Supplement fertilizer supplies for meeting the nutrient needs of crops.
• Add 20 – 200 kg N/ha (by fixation) under optimum conditions and solubilise/mobilise
30-50 kg P2O5/ha.
• They liberate growth promoting substances and vitamins and help to maintain soil
fertility.
• They suppress the incidence of pathogens and control diseases.
• Increase the crop yield by 10-50%. N2 fixers reduce depletion of soil nutrients and
provide sustainability to the farming system.
• Cheaper, pollution free and based on renewable energy sources.
• They improve soil physical properties, tilth and soil health.

9.6.6 Rhizobium
Rhizobia are soil bacteria, live freely in soil and in the root region of both leguminous and
non-leguminous plants. However, they enter into symbiosis only with leguminous plants, by
infesting their roots and forming nodules on them. Non legume nodulated by Rhizobia is
Trema or Parasponia sp. The nodulated legumes contribute a good deal to the amount of N2
fixed in
the biosphere, (50-200 kg N/ha) varied with crops. The nitrogen fixation by clover and
cowpea is 130 kg N/ha and 62-128 kg N/ha respectively. Beijerinck first isolated and
cultivate a microorganism from the roots of legumes in 1888 and he named this as Bacillus
radicola and latter modified as Rhizobium. Legume plants fix and utilise this N by working
symbiotically with Rhizobium in nodules on their roots. The host plants provide a home for
bacteria and energy to fix atmospheric N2 and in turn the plant receives fixed N2 (as protein).

9.6.7 Biotechnological applications for horticulture

The requirement of fruits and vegetables is increasing proportionally with the increasing
population in the country. How do we keep horticultural production on par with the
burgeoning population? Although conventional plant breeding techniques have made
considerable progress in the development of improved varieties, they have not been able to
keep pace with the increasing demand for vegetables and fruits in the developing countries.
Therefore, an immediate need is felt to integrate biotechnology to speed up the crop
improvement programmes. Biotechnological tools have revolutionized the entire crop
improvement programmes by providing new strains of plants, supply of planting material,

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more efficient and selective pesticides and improved fertilizers. Many genetically modified
fruits and vegetables are already in the market in developed countries. Modern
biotechnology encompasses broad areas of biology from utilization of living organisms or
substances from those organisms to make or to modify a product, to improve plant or animal
or to develop micro-organisms for specific use. It is a new aspect of biological and
agricultural science which provides new tools and strategies in the struggle against world’s
food production problem. The major areas of biotechnology which can be adopted for
improvement of horticultural crops are; tissue culture, genetic engineering, molecular
diagnostics, molecular markers and development

9.6.8 Biotechnological applications for Forestry

At present, most of the planting material for different afforestation programscomes from
sources that are genetically diverse and that give poor yields. Therefore,in order to enhance
productivity, it is essential to have a highly efficient productiontechnology including high
yielding certified planting material. In this context, tissueculture and other biotechnological
tools can play an important role in boosting productivity. Tissue culture technology, together
with mycorrhizae, nitrogen fixing microorganisms and institutional support, etc. have been
used to enhance the productivity offorest trees in the United States, Brazil, Japan and some
West European countries.Success has also been obtained in the case of orchids, ornamentals
and a number ofvegetable and fruit species. Species of strawberry and apple are now
produced commercially by tissue culture. In India, the technique of raising plants through
tissueculture has been perfected for a few tree species, e.g., Bamboo, Dalbergia, Eucalyptus,
Leucaena, Prosopis, Santalum, Sesbania and elites of Tectona. In order to achieve the
quantum jump in the production of biomass for fuel, fodder, timber and industrial woods
using the new tools of biotechnology, the following species have been identifiedfor mass
propagation through tissue culture: Acacia nilotica, Alnus nepalensis, Hardwickia binata,
Madhuca latifolia, Prosopis cineraria, Tamarindus indica, Dendrocalamus strictus, Bambusa
arundinacea, Bambusa vulgaris, Tectolla grandis, Shorea robusta, Dalbergia latifolia,
Santalum album, Populus deltoides.
It is necessary that greater emphasis be given to the following three areas foroverall
applications of biotechnology towards the enhancement of biomass production, (1).
Improvement ill overall growth of forests. This involves improvement in regeneration
techniques, ensuring better survival of young saplings, development of improved genetic
stocks and reduction in the damage caused by fire, pests, etc. Plantingwith genetically
improved stock alone can increase the timber yield per hectare byabout 25 percent over
current rates of growth, (2). Advances ill the products manufactured from forest resources.
Here, the potential is limited only by the researchers' vision. A substantial reduction in the
susceptibility of wood to decay could improve the useful life of products ranging from
thoseused in construction to utility poles.
(3). Understanding the impacts of non-forestry related activities on forest. More research is
required to understand the effect of acid rain on thegrowth and productivity of the forests.
This work will lead to solutions to mitigate theimpact of this by-product of industrial
growth.Organisational integration at the level of the individual scientist, department and
organisation must be taken into account if biotechnology is to have any realisticapplication in

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enhancing biomass production. Optimally efficient research integrationis determined, in part,

by the organisational setting involved and the technologies. employed. If research and
product development capabilities arc not contained withinthe same organisation, they must be
established through licensing arrangements,collaborative research or research network.The
integration of new technologies can be achieved through multi-disciplinaryteams whose
objectives are in agreement with those of conventional plant breeders.Collaborative research
also necessitates the support of plant breeders for evaluationand testing. In the following
areas, both applied as well as the basic aspects of biotechnology need to be integrated with
conventional methods, in order to make an impacton forestry:
(i) Mass propagation of elite plants through tissue culture
(ii) Scaling up the production of artificial seeds
(iii) Cryopreservation of the gene pools of elite trees
(iv) Exploiting somaclonal variants for tree improvement
(v) Microspore and another culture for fixation of heterosis
(vi) Development, in some important tree species of a genetic transformation system using
agrobacterium and regeneration of transformed plantlets by tissueculture. Genetic
manipulation of trees through protoplast fusion or somatic hybridisation
(vii) Large-scale field evaluation of tissue culture raised plants.

9.6.9 Biotechnological applications for medicines

Biotechnology has a variety of applications in the field of medicine. Some of the
biotechnology applications in medicine include the following:
9.6.9.1 Recombinant Insulin
Insulin is required by diabetic patients to remove excess sugar from the blood. Diabetic
patients have a very low level of insulin or no insulin produced by the body. Therefore, they
need external insulin to control blood glucose levels. Later it was discovered that the insulin
produced by the pancreas of the pigs can be used by humans. But there were not enough pigs
to provide the quantities of insulin required. This led to the cloning of the human insulin
gene.The specific gene sequence that codes for human insulin were introduced in E.coli
bacteria. The gene sequence altered the genetic composition of the E.coli cells. Within 24
hours several E.coli bacteria containing the recombinant human insulin gene were produced.
The recombinant human insulin was isolated from E.coli cells.
9.6.9.2 Gene Therapy
Gene Therapy holds the most promising answer to the problem of genetic diseases. Gene
therapy is used to treat genetic disorders usually by the insertion of a normal gene or correct
gene for the defective or inactive gene into an individual with the help of vectors such as
retrovirus, adenovirus, and herpes simplex virus.The normal gene replaces the defective or
inactive gene and carries out its functions. The therapy has the highest chances of developing
a permanent cure if introduced in the earliest stages of life.
9.6.9.3 Molecular Diagnosis
Medical diagnosis is another application of biotechnology in the health sector. Many times,
the pathogen concentration increases by the time the disease is diagnosed. Hence, early
diagnosis and knowledge of pathophysiology are essential for an effective cure. This can be

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achieved with the help of techniques such as Recombinant DNA Technology, Polymerase
Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), etc.
9.6.9.4 Pharmacogenomics
Pharmacogenomics has led to the production of drugs that are best suited to an individual’s
genetic makeup. It can be applied in diseases such as cancer, depression, HIV, asthma, etc.
9.6.9.5 Edible Vaccines
Vaccines are obtained by animals and cell cultures. These vaccines contain inactivated
pathogens.The transgenic plants can produce antigens that can be used as edible vaccines.
Antigenic proteins from several pathogens can be expressed in plants such as tomato and
banana. Transgenic sugarbeet can treat foot and mouth disease of animals, transgenic banana
and tomato can cure diseases such as cholera and hepatitis B.

9.7 BIOTECHNOLOGICAL APPLICATIONS FOR INDUSTRY

Industrial biotechnology includes modern application of biotechnology for sustainable
processing and production of chemical products, materials and fuels. Biotechnological
processing uses enzymes and microorganisms to produce products that are useful to a broad
range of industrial sectors, including chemical and pharmaceutical, human and animal
nutrition, pulp and paper, textiles, energy, materials and polymers, using renewable raw
materials.Use of biotechnology to substitute existing processes makes many of these
industries more efficient and environmentally friendly, contributing to industrial
sustainability in various ways. This paradigm change involves various areas, ranging from the
most known ones, such as pharmaceutical and agricultural, to production of materials such as
biopolymers and also bioplastics.Industrial biotechnology can produce the same results as the
petrochemical industry, but using biological catalysts instead. Application of the state of the
art of a vast range of scientific disciplines to industrial biotechnology, namely biochemistry,
microbiology, genomics, proteomics, bioinformatics, systems biology and process
engineering is the foundation for leveraging the rapid, specialized and competitive growth of
the sector, based on biocatalysts that enable high productivity, performance and stability.With
the adoption of industrial processes based on biotechnology, metabolic engineering has
become an increasingly important subject. The goal of metabolic engineering is to maximize
the production of compounds that are of industrial interest in microorganisms that act within
this context as cell factories through their genetic manipulation.According to a recent OECD
study (The Bioeconomy to 2030: designing a policy agenda,
http://www.oecd.org/futures/bioeconomy/2030), the industrial applications of biotechnology
in 2030 will be responsible for 39% of the economic value generated by Biotechnology,
which illustrates the healthy investment in research and development expected in this area.

9.8 FERMENTATION
Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. It occurs in yeast
and bacteria, and also in oxygen-starved muscle cells, as in the case of lactic acid fermentation.
Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth
medium, often with the goal of producing a specific chemical product like enzyme, vaccines,
antibiotics, food product/additive etc. French microbiologist Louis Pasteur is often remembered for

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his insights into fermentation and its microbial causes. The science of fermentation is known as
zymology
Fermentation takes place in the lack of oxygen (when the electron transport chain is
unusable) and becomes the cell’s primary means of ATP (energy) production. It turns NADH
and pyruvate produced in the glycolysis step into NAD+ and various small molecules
depending on the type of fermentation. In the presence of O2, NADH and pyruvate are used
to generate ATP in respiration. This is called oxidative phosphorylation, and it generates
much more ATP than glycolysis alone. For that reason, cells generally benefit from avoiding
fermentation when oxygen is available, the exception being obligate anaerobes which cannot
tolerate oxygen.
The first step, glycolysis, is common to all fermentation pathways:
C6H12O6 + 2NAD+ 2ADP ++ 2Pi → 2 CH3COCOO− + 2 NADH + 2 ATP + 2 H2O +2H+
The pyruvate is CH3COCOO−. Pi is inorganic phosphate. Two ADP molecules and two Pi
are converted to two ATP and two water molecules via substrate-level phosphorylation. Two
molecules of NAD+ are also reduced to NADH. In oxidative phosphorylation the energy for
ATP formation is derived from an electrochemical proton gradient generated across the inner
mitochondrial membrane (or, in the case of bacteria, the plasma membrane) via the electron
transport chain. Glycolysis has substrate-level phosphorylation (ATP generated directly at the
point of reaction).
Humans have used fermentation to produce food and beverages since the Neolithic age. For
example, fermentation is used for preservation in a process that produces lactic acid as found
in such sour foods as pickled cucumbers, kimchi and yogurt, as well as for producing
alcoholic beverages such as wine and beer. Fermentation can even occur within the stomachs
of animals, such as humans.

9.8.1 Definitions of Fermentation

To many people, fermentation simply means the production of alcohol: grains and fruits are
fermented to produce beer and wine. If a food soured, one might say it was 'off' or fermented.
Here are some definitions of fermentation. They range from informal, general usage to more
scientific definitions.
1. Preservation methods for food via microorganisms (general use).
2. Any process that produces alcoholic beverages or acidic dairy products (general use).
3. Any large-scale microbial process occurring with or without air (common definition used
in industry).
4. Any energy-releasing metabolic process that takes place only under anaerobic conditions
(becoming more scientific).
5. Any metabolic process that releases energy from a sugar or other organic molecules, does
not require oxygen or an electron transport system, and uses an organic molecule as the final
electron acceptor (most scientific).

9.8.2 Examples of Fermentation

Fermentation does not necessarily have to be carried out in an anaerobic environment. For
example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to
aerobic respiration, as long as sugars are readily available for consumption (a phenomenon

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known as the Crabtree effect). The antibiotic activity of hops also inhibits aerobic metabolism
in yeast. Fermentation react NADH with an endogenous, organic electron acceptor. Usually
this is pyruvate formed from the sugar during the glycolysis step. During fermentation,
pyruvate is metabolized to various compounds through several processes:
1. Ethanol fermentation, aka alcoholic fermentation, is the production of ethanol and carbon
dioxide
2. Lactic acid fermentation refers to two means of producing lactic acid:
(a) Homolactic fermentation is the production of lactic acid exclusively
(b) Heterolactic fermentation is the production of lactic acid as well as other acids and
alcohols.
Sugars are the most common substrate of fermentation, and typical examples of fermentation
products are ethanol, lactic acid, carbon dioxide, and hydrogen gas (H2). However, more
exotic compounds can be produced by fermentation, such as butyric acid and acetone. Yeast
carries out fermentation in the production of ethanol in beers, wines, and other alcoholic
drinks, along with the production of large quantities of carbon dioxide. Fermentation occurs
in mammalian muscle during periods of intense exercise where oxygen supply becomes
limited, resulting in the creation of lactic acid.

9.9 SUMMARY
The biotechnology is an interdisciplinary branch of science that deals with biochemistry,
microbiology, genetic engineering for mankind. Biotechnology offers various aspect of
application in agriculture, horticulture, forestry, medical and industries. The trained
biotechnologist can help the society by developing biofertilizers in agriculture, development
of genetical modified plants and production of value-added products using industrial
microbiology. In due to the development of the next generation high-throughput technologies
new branch emerged as bioinformatics. Bioinformatics mainly deals with the genomic,
transcriptomics and proteomics datasets generated from the living systems.

9.10 BIBLIOGRAPHY
Maithani, D., Sharma, A., Gangola, S., Choudhary, P., Bhatt, P., 2022.Insights into
applications and strategies for discovery of microbial bioactive metabolites.
Microbiological Research. 261, 127053.
Karim A, Gerliani N, Aïder M. 2020. Kluyveromyces marxianus: An emerging yeast cell
factory for applications in food and biotechnology. Int J Food Microbiol.333:108818. doi:
10.1016/j.ijfoodmicro.2020.108818.
International Wheat Genome Sequencing Consortium (IWGSC). 2018. Shifting the limits in
wheat research and breeding using a fully annotated reference genome. Science. 2018 Aug
17; 361(6403):eaar7191. doi: 10.1126/science.aar7191.

Suggested reading
Bhatt, P., 2022. Industrial applications of microbial enzymes. ISBN:9781003202998. CRC
Press publisher, Boca Raton. https://doi.org/10.1201/9781003202998.

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9.11 TERMINAL QUESTIONS

Q1. Discuss the role of biotechnology in agriculture in detail
Q2. Write notes on following
i. Genetic Engineering
ii. Role of biotechnology in horticulture
iii. Prevention of genetic disorders
iv. Treatment of diseases and genetic disorders
Q3. What are the varieties of applications in the field of medicine?
Q4. What are the applications of biotechnology in Industry? Discuss in detail.
Q5. Define fermentation and explain its process along with the examples.

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