ARBA MINCH UNIVERSITY

COLLEGE OF NATURAL SCIENCES

DEPARTMENT OF BIOLOGY

Introduction to Biotechnology(Biot.3002)

Prepared by: Samson

Tizazu(MSc)
 Biotechnology : Definitions and Scope

1.1. Definitions of Biotechnology

 The term biotechnology is a fusion of biology and technology.
 It is derived from two words
o Bio = Life
o Technology = Application of tools and methods for
production
 The term was used before the 20thC for traditional activities
such as making dairy products such as cheese and curd, as
well as bread, wine, beer, etc
 But all of these could be considered as traditional
biotechnology
 Biotechnology discipline emerged at the end of 20th century
which integrate biology with technology
 Therefore, biotechnology is concerned with exploitation of
biological components for production of useful products. 2
 Cont…
 Defined by different organization in different ways:
 Broadly, defined as “the development and utilization of
biological process, forms and systems for obtaining maximum
benefits to man and other forms of life”
 Biotechnology is the application of scientific and engineering
principles to the process of materials by biological agents to
provide goods and services [The Organization for Economic
Coorporation And Development(OECD),1981].
 The integrated use of biochemistry, microbiology and
engineering sciences in order to achieve technological
applications of the capabilities of microorganisms, culture
tissue, cells [ The Europian Federation Of Biotechnology
(EFB), 1981; O’sullivan, 1981]
 The application of biochemistry , biology , microbiology
and chemical engineering to industrial process and
products and on environment [ International Union Of Pure
And Applied Chemistry (IUPAC), 1981]
 Cont…
 According to United States National Science Academy,
Biotechnology is “the controlled use of biological agents
like cells or cellular components for beneficial use”.

 British Biotechnologists defined Biotechnology as “the

application of biological organisms, systems or processes
to manufacturing and service industries”.
 UN Convention on Biological Diversity (CBD)
‘’any technological application that uses biological
systems, living organisms, or derivatives thereof, to make
or modify products or processes for specific use.’’
 More generally, biotechnology can be defined as “the use
of living organisms, cells or cellular components for the
production of compounds or precise genetic improvement
of living things for the benefit of man”.
 1.2 History and Scope of Biotechnology
 The biotechnology date back to the time (around 6000
BC)
 when the yeast was first used to produce beer and wine
and the bacteria were first used to prepare yogurt.
 The biotechnology revolution began in 1970s and early
1980s when the scientist understood the genetic
constitution of living organism.
 Strong foundation of genetic engineering and modern
biotechnology was laid down by Cohen and Boyer in 1973
 Successfully they introduced the desired gene of one
organism into another and clone the new gene.
 The development of Biotechnology, in the first half of 20th
century associated with the field of applied :
→microbiology = production of penicillin,
→industrial fermentations = production of organic solvents
etc
 Cont…
 The development of biotechnology also closely linked
with the advancement made in molecular biology
 Biotechnology consists of a gradient of technologies,
ranging from the long-established and widely used
techniques of traditional biotechnology to novel and
continuously evolving modem biotechnology
techniques

Classical and Modern Biotechnology

 There are no differences in the principles but; the
technological advancement of utilizing living cells for
A. Classical Biotechnology

 It is also called kitchen technology developed by our ancestor using

fermenting bacteria
 Sumerians and Babylonian (6000BC) – were drinking the beer.
 Egyptians (4000 BC)- were baking leavened bread.
 Beer , wine ,cheese , and many foods have been produced using
traditional biotechnology.
 Includes the process that are based on the natural capabilities of
microorganism.
 Is basically a microbial-based fermentation process
 In short, it is a hybrid of fermentation and biochemical engineering.
 Industries linked to the fermentation technology had grown
tremendously because of the high demand for various
chemicals such as ethanol, butanol, glycerine, acetone, etc.
 B. Modern Biotechnology
 It is mainly based on genetic engineering/recombinant
DNA technology (rDNA) and hybridoma technology in
addition to bioprocess technology.

 rDNA technology: is the main tool to produce

genetically-modified organisms (GMO), including
plants , animals, & microbes, but also to address the
fundamental questions in life sciences. e.g. Golden rice

 In fact, modern biotechnology began when

recombinant human insulin was produced and
marketed in the United States in 1982.
 The following are the basic steps
involved in the process of the rDNA
technique for gene cloning.
o Extraction of DNA and
isolation of gene of interest
o Generating DNA fragments
o Insertion of DNA in to
vectors
o Transformation of host cells
o Selection of recombinant
cells
 e.g. Production of insulin by rDNA
technology

9
 Cont…
 The process of fermentation for the preparation and
manufacturing of products such as alcohol, beer, wine,
dairy products, various types of organic acids such as
vinegar, citric acid, amino acids, and vitamins can be
called classical biotechnology or traditional
biotechnology.
 Modern biotechnology is similar to classical
biotechnology in utilizing living organisms.
 It is not modern in the sense of using various living
organisms, but in the techniques for doing so (genetic
manipulation).
 The introduction of a large number of new techniques
has changed the face of classical biotechnology forever.
 Applications of Biotechnology
 Agricultural Applications
 Production of
o Transgenic animals for:
• increased milk production and growth rate,
• resistance to diseases, etc…
o Transgenic plants for:
• Insect and herbicide resistance
• Virus protection
• Increased nutritional quality and yield
• Production of useful products
 Cont…
 In vitro fertilization- for rapid multiplication of
animals
 Rapid multiplication of plants through plant tissue
culture
 Environmental Applications
 Sewage treatment
 Bioremediation: (e.g. degradation of petroleum and
management of oil spill)
 Biopestcides: biological control of plant diseases &
insect pests
 Bio-fertilizers,
 Bioleaching, etc
 Medical Applications

 For production of:

o antibiotics,
o insulin,
o growth hormone,
o Interferon (used against certain cancers and
viral infections)
o clotting factor VIII (used to treat hemophilia)
o vaccines,
o probes for infectious and gene therapy and so on.
 Industrial Applications
 For production of:
o proteins,
o enzymes,
o Antibiotics
o Metabolites
END!
 Chapter 2
Recombinant DNA technology
1. Restriction enzymes
1.2. Genetic Engineering / Recombinant DNA
Technology
The production of a unique DNA molecule by joining
two or more different DNA fragments together
 It is the art of cutting and pasting genes.
 is one of the recent advances in biotechnology,
which was developed by two scientists (Boyer and
Cohen in 1973)
 It encompasses a number of methodologies or tools
to construct new combinations of DNA
(recombinant DNA or rDNA).
 The rDNA molecule constructed thus can be
introduced into an appropriate host cell,
 It can be multiplied to generate many copies in the
host cell => This is known as gene cloning or DNA
cloning.
 Cont…
 The goal of rDNA tech/gene cloning is to generate large amounts
of pure DNA that can be manipulated and studied.

 rDNA technology is also used for preparing human therapeutic

proteins, vaccines, and diagnostic proteins is by hybridoma
technology.
 The first hybridoma experiments were carried out in 1975.
 In hybridoma technology, a B-lymphocyte secreting antibody
against a specific antigen is fused with a myeloma cell.
 The resulting (a cancerous B-lymphocyte) cell, if injected into a
mouse’s abdomen or if cultured in a bioreactor by applying
bioprocess technology, will grow and divide indefinitely, producing
large quantities of the antibody, which can then be harvested
 In agriculture, rDNA technology can be used to produce new
varieties of crop plants with improved agricultural and nutritive
qualities.
 Transgenic plants, which are resistant to biotic and abiotic
stresses such as salinity, drought, and disease, have been
produced
 The following are the basic steps
involved in the process of the rDNA
technique for gene cloning.
o Extraction of DNA and
isolation of gene of interest
o Generating DNA fragments
o Insertion of DNA in to
vectors
o Transformation of host cells
o Selection of recombinant
cells
 e.g. Production of insulin by rDNA
technology

19
 20
Fig. The basic steps in gene cloning
Fig. Using a restriction
enzyme and DNA
ligase to make
recombinant DNA

21
 1.4. Restriction Endonucleases
 Endonucleases are enzymes that break internal phosphodiester bonds
within a DNA molecule.
 are DNases that recognize specific nucleotide sequences and cut dsDNA in
a site.
 These endonucleases can be non-specific or restriction
Non specific endonucleases - cleave DNA at any internal
phosphodiester bond with the end product being a
mononucleotide or very short oligonucleotides ( e.g. DNase I)
Restriction endonucleases - on the other hand cleave dsDNA
only at a limited number of specific recognition sites.
 Therefore, RE give rise to discrete fragments of defined length and
sequence.
 It was discovered by Kelly and Smith in 1970 from Haemophilus influenzae.
Example, EcoRI G’AATTC
GTTAA’G
 Source of Restriction Endonucleases (RE)
 Is extracted and purified from bacteria.
 The bacterium uses the enzyme to protect itself from
bacteriophage (viral) infection by restricting its
replication.

 RE recognize specific 4- 8 bp sequences in the

incoming DNA called restriction sites & then cleave
both DNA strands at this site.

 Bacterium protects its own DNA from RE by

methylating those specific sequences that can be
recognized by RE.
 The additional methyl group blocks the degradative
enzymes.
 A/ NOMENCLATURE
 Restriction endonucleases are named for the organism in
which they were discovered, using a system of letters and
numbers.
 was proposed by Smith and Nathans in 1973
 Features of the name:
 RE are named type of bacteria in which the enzyme is
derived.
Example1: EcoRI
o Is isolated from Escherichia (E) coli (co),
o Strain Ry13 (R) and
o It is the first (I) endonuclease to be discovered from the organism.
Example 2: HindIII
o Isolated from Haemophilus (H) influenzae (in),
o Strain Rd (d) and
o It is the third (III) endonuclease to be discovered from the organism.
 B/ RECOGNITION SEQUENCES
 Each RE always cuts at the same recognition
sequences.
 Many restriction sequences are palinromic sites.
E.g. EcoRI 5’GAATTC3’
3’CTTAAG5’
 Read the same in opposite direction (e.g. MADAM)
 Most type II REs cleave DNA in their specific
recognition sequences.

 Recognition sequences are distributed throughout the

genome.

 Thus, RE digestion results in specific DNA fragments

that can be resolved by gel electrophoresis based on
their size.
26
 C). BLUNT END / FLUSH END CUTTERS

 The RE cuts the dsDNA in the middle of the

recognition sequence
 have non-sharp, even or flash end (not overhanging)
E.g. SmaI, PvuII and AluI

27
 D). STICKY / STAGGERED CUTTERS

 The two DNA strands are not cut at the same

position
 Produce fragments that have a single-stranded “tail”,
overhanging at both ends.
 The protruding ends are also called sticky ends /
cohesive ends.
 Base pairing b/n the ends can stick the DNA
molecule back together.

28
 An important feature is that different RE with
different recognition sequences may produce
complementary sticky ends.
Fragments of DNA produced by cleavage with either of
these enzymes can be joined together as each
fragment carries a complementary sticky end.

29
30
31
Examples of Type II RE used in rDNA
Experiments

32
33
 CHAPTER- TWO
2. BIOTECHNOLOGY IN HEALTH
CHAPTER OUTLINES

2.1. Hybridoma Technology and Monoclonal Antibodies

2.2. Application of Biotechnology in Medicine
2.2.1. Diagnosis
2.2.2. Vaccine Production
2.2.3. Gene Therapy
2.2.4. Organ/ Tissue Transplantation
2.2.4. Forensic Medicine
2.2.5. Stem Cell Research and Its Potential in
Medicine
2.2.5. Protein Based Drugs

36
 2.1. HYBRIDOMA TECHNOLOGY AND MONOCLONAL
ANTIBODIES DISCOVERY

 In the 1970s, the B-cell cancer myeloma was known, and it

was understood that these cancerous B-cells all produce a
single type of antibody. This was used to study the structure
of antibodies, but it was not possible to produce identical
antibodies specific to a given antigen.
 The process of producing monoclonal antibodies invented by
Georges Köhler and César Milstein in 1975; they shared the
Nobel Prize in Physiology or Medicine in 1984 for the
discovery.
 The key idea was to use a line of myeloma cells that had lost
their ability to secrete antibodies, and come up with a
technique to fuse these cells with healthy antibody
producing B-cells.

37
 Hybridoma Technology
 Hybridoma technology- is the production of a hybrid
cell a fusion product of an antibody-secreting immune cell
and an immortal or cancerous immune cell.
 Its application in the continuous synthesis of the
respective antibody industrially for medicinal
application.
 Hybridoma is created by fusing two cells, a secreting cell
from the immune system and a long-lived (immortal)
cancerous immune cell, within a single membrane.
 The resulting hybrid cell can be cloned, producing many
identical cells.
 Each of these daughter clones can secrete the immune
cell product over a long period of time.
38
  Antibody
 Antibody- is kinds of normally occurring protein molecules
that are produced in the body cells called lymphocytes and
that act primarily as a defense against invasion by foreign
substances.
 are Y-shaped proteins called immunoglobulins (Ig) and
are made only by B cells.
 An important component of the immune system, antibodies
are found in the blood of all vertebrates, in the fraction of the
blood called gamma globulin.
 The synthesis of antibodies is initiated when a foreign
substance, referred to as an antigen, enters the body.
 Lymphocyte cells respond to the foreign substance by
making an antibody.
 Common antigens are the protein components of bacteria
and viruses. 39
 There are five different kinds of antibodies.
 They are IgG, IgM, IgA, IgD, and IgE
 When a new antigen comes into the body, it binds to
the B-cell, which is already making an antibody that
matches the antigen
 IgM- is the first antibody made by newborns and the
first made during an infection
 IgG - is the predominant antibody in serum; it is
made on the second exposure to an antigen.
 IgE- is associated with allergy.
 IgA- is found in saliva and mother's milk.
 The role of IgD is not known.
40
  Monoclonal
 Monoclonal antibodies- are antibodies that
Antibodies
are identical because they were produced by one
type of immune cell (B cell), all clones of a single
parent cell.
 Given any substance, it is possible to create
monoclonal antibodies that specifically bind to that
substance; they can then serve to detect or purify
that substance.
 This has become an important tool in biochemistry,
molecular biology and medicine.

41
  ANTIBODIES
POLYCLONAL MONOCLONAL
Derived from different B Derived from a single B
Lymphocytes cell lines cell clone

mAb offer reproducible,

Batch to batch variation predictable & potentially
affecting Ab reactivity & inexhaustible supply of
titre Ab with exquisite
specificity
NOT Powerful tools for Enable the development
clinical diagnostic tests of secure immunoassay
systems.

42
 HYBRIDOMA TECHNOLOGY

43
 Cont’d…
Step 1: - Immunization of mice & selection of
mouse
donor for generation of hybridoma
cells.
ANTIGEN ( Intact cell/
Whole cell
membrane/ micro-
organisms ) +
ADJUVANT
(Emulsification)
Ab titre reached in
Serum
Spleen removed
(Source of cells)
44
 Cont’d…
Step 2: - Screening of mice for antibody
production
After several
weeks of
immunization
Serum Antibody Titre
Determined
(Technique: ELISA)
Titre too Titre
low High 2
BOOST BOOST weeks
(Pure (Pure
antigen) antigen)
45
 Cont’d…
Step 3: - Preparation of myeloma cells

+ 8 - Azaguanine
Myeloma Cells
Immortal Tumor Of
Lymphocytes

Myeloma Cells
HGPRT-
High Viability & Rapid
Growth 46
 Cont’d…
Step 4: - Fusion of myeloma cells with immune
spleen
cells & selection of hybridoma cells
PEG
FUSION
SPLEEN MYELOMA
CELLS Feeder Cells CELLS
Growth Medium

1. Plating of Cells
in HAT selective
HYBRIDOMA CELLS Medium
ELISA PLATE 2. Scanning of
HAT Medium Viable
Hybridomas

47
 Cont’d….
Step 5: - Cloning of hybridoma cell lines by “
limiting
dilution” or expansion

A. Clone Each +ve Culture

B. Test Each Supernatant for

Antibodies

C. Expand +ve Clones

Tissue Mouse
Culture As
Method cites
Metho
d
48
 HGPRT (hypoxanthine-guanine phosphoribosyl
transferase)
 HAT (hypoxanthine-aminopterin-thymine) medium
 ELISA (enzyme-linked immunosorbent assay) is a
highly sensitive method for the detection of
antibodies or soluble antigens in sera and other
body fluids.

49
 Applications of Monoclonal Antibodies

Diagnostic Applications
 Biosensors & Microarrays
Therapeutic Applications
 Transplant rejection
 Cardiovascular disease
 Cancer
 Infectious Diseases
 Inflammatory disease
Clinical Applications
 Purification of drugs, Imaging the target
Future Applications
 Fight against Bioterrorism
50
2.2. Application of Biotechnology in
Medicine
2.2.1. Diagnostics
 The success of modern medicine
depends on the detection of specific
molecules e.g.
 Viruses
 Bacteria

 Fungi
 Proteins
51
 Characteristics of a Detection
System
 A good detection system should have 3 qualities:
 Sensitivity
 Specificity
 Simplicity
Sensitivity: means that the test must be able to
detect very small amounts of target even in the
presence of other molecules.
Specificity: the test yields a positive result for the
target molecule only.
Simplicity: the test must be able to run efficiently
and inexpensively on a routine basis.
52
 A. Immunological Diagnostic
 System
Immunological diagnostic procedures are often used to:
 Test drugs
 Monitor cancers
 Detect pathogens
 ELISA (Enzyme Linked Immunosorbent Assay)
 This involves the reaction of an antibody with an
antigen and a detection system to determine if a
reaction has occurred.

53
 ELISA Involves:
 Binding of the test molecule or organism to a solid
support. e.g. micro titer plate.
 Addition of a specific antibody (primary antibody) which
will bind to the test molecule if it is present.
 Washing to remove unbound molecules.
 Addition of secondary antibody which will bind to the
primary antibody.
 The secondary antibody usually has attached to it an
enzyme. e.g. alkaline phosphatase.
 Wash to remove unbound antibody.
 Addition of a colorless substrate which will react with the
secondary antibody to give a color reaction which
indicates a positive result.
54
ELISA

55
ELISA Lab/Detection of HIV

56
Detection of
HIV

57
B. DNA Diagnostic Systems

DNA Diagnostic Systems include:

1) DNA Hybridization

2) PCR

3) DNA fingerprinting

4) RAPD (Random Amplified Polymorphic

DNA)

5) Biosensors
58
 1. DNA
 Bacterial and viral pathogens may be pathogenic
Hybridization
because of the presence of specific genes or sets of
genes.
 Genetic diseases often are due to mutations or
absence of particular gene or genes.
 These genes (DNA) can be used as diagnostic tools.

 This involves using a DNA probe during DNA

hybridization.
 What is a DNA probe?
 How does DNA hybridization work?
59
60
 Cont’d…
 For DNA hybridization:
 A probe is needed which will anneal to the target
nucleic acid.
 Attach the target to a solid matrix ( e.g. membrane).
 Denaturation of both the probe and target.
 Add the denatured probe in a solution to the target.
 If there is sequence homology between the target and
the probe, the probe will hybridize or anneal to the
target.
 Detection of the hybridized probe e.g. by
autoradiography, chemiluminsence or colorimetric.
61
 Detection of Malaria
 Malaria is caused by the parasite of the genus
Plasmodium.
What kind of organism is P. falciparum?
 The parasite infects and destroys red blood cells (RBCs).
 Symptoms include fever, rashes and damage to
brain, kidney and other organs.
Current treatment involves microscopic observations
of blood smears, which is labor intensive.
Other methods (e.g. ELISA does not differentiate
between past and present infection).
Q/ Why?

62
 Cont’d…
 A DNA diagnostic system would only
measure current infection. (Why?)
o The procedure involves:
 A genomic library of the parasite was
screened with probes for parasitic DNA.
 The probes which hybridized strongly were
tested further.
 The probes were tested for their ability to
hybridize to other Plasmodium species which
do not cause malaria and to human DNA.

63
 Cont’d…
 Probes which hybridized to P. falciparum only could
be used as a diagnostic tool.
 The probe was able to detect 10 pg of purified DNA
or 1 ng of DNA in blood smear.
 Other DNA probes were developed for the following
diseases:
 Salmonella typhi (food poisoning)

 E. coli (gastroenteritis)
 Trypanosoma cruzi (chagas’ disease)
64
 2) Polymerase Chain Reaction
 PCR uses 2 sequence specific oligionucleotide
primers to amplify the target DNA.
 The presence of the appropriate amplified size
fragment confirms the presence of the target.
 Specific primers are now available for the
detection of many pathogens including bacteria
(E. coli, M. tuberculosis), viruses (HIV) and
fungi.

65
  Using PCR to Detect for
HIV
RT-PCR (Reverse Transcriptase PCR).
HIV has a ssRNA genome.
 Lyse plasma cells from the potentially infected
person to release HIV RNA genome.
 The RNA is precipitated using isoproponal.
 Reverse transciptase is used to make a cDNA copy
of the RNA of the virus.
 The Reverse transcriptase converts RNA into DNA.
 This cDNA is used as a template to make dsDNA.

66
 Using PCR to Detect for HIV
 Specific primers are used to amplify a 156 bp
portion of the HIV gag gene.
 Using standards the amount of PCR product
can be used to determine the viral load.
 PCR can also be used as a prognostic tool to
determine viral load.
 This method can also be used to determine the
effectiveness antiviral therapy.
67
 3) DNA Fingerprinting
(RFLP)
 RFLP = Restriction Fragment Length Polymorphism
 Regular fingerprinting analyses phenotypic
traits.
 DNA fingerprinting analyses genotypic traits.

 DNA fingerprinting (DNA typing) is

used to characterize biological samples
e.g.
 In legal proceedings to identify suspects and
clear others. 68
 Cont’d…
The procedure involves:
 Collection of sample (e.g. hair, blood, semen, and
skin).
 Examination of sample to determine if there is enough
DNA for the test.
 The DNA is digested with restriction enzymes.
 Digested DNA is separated by agarose gel
electrophoresis.
 DNA is transferred by Southern blotting to a membrane.
 Membrane is hybridized with 4-5 different probes.
 Detection of hybridization.
69
  Microsatellite DNA
 After hybridization the membranes are stripped and
reprobed.
 The probes used are human microsatellite DNA.
 These sequences occur in the human genome as
repeated sequences.
E.g. ATTAG….ATTAG….ATTAG….
 The length of the repeat is 9-40 bases occurring 10-
30 times.
 The microsatellites have different length and
numbers in different individuals.
 The variability is due to either a gain or lost of
repeats during replication.

70
 Cont’d…
 These changes do not have any biological effect
because the sequences do not code for any protein.
 An individual inherit one microsatellite from each
parent.
 The chance of finding two individuals within the
same population with the same DNA fingerprint is
one in 105 - 108.
 In other words an individuals DNA fingerprint is
almost as unique as his or her fingerprint.
71
 DNA
Fingerprinting

72
 4) Random Amplified Polymorphic DNA
(RAPD)
 Another method widely used in characterization of
DNA is RAPD.
 RAPD is often used to show relatedness among DNA
populations.
 In this procedure arbitrary (random) primers are
used during PCR to produce a fingerprint of the
DNA.
 A single primer is used which must anneal in 2 places
on the DNA template and region between the
primers will be amplified.
73
 Cont’d…
 The primers are likely to anneal in many places on the
template DNA and will produce a variety of sizes of
amplified products.
 Amplified products are separated by agarose gel
electrophoresis and visualized.
 If the samples have similar genetic make up then the
pattern of bands on the gel will be similar and vice
versa.
 This procedure is widely used to differentiate between
different cultivars/varieties of the same plant.
 Issues to consider when using this procedure include
reproducibility, quality of DNA, and several primers may
have to be used.
74
RAPD

75
 5) Bacterial
Biosensors
 Bacterial sensors can be used to test for environmental
pollutants.
 Bacteria with bioluminescent are good candidates for sensors.
 In the presence of pollutants the bioluminescent decreases.
 The structural genes (luxCDABD) encodes the enzyme for
bioluminescent was cloned into the soil bacteria
Pseudomonas fluorescens.
 The cells that luminescence to the greatest extent and grew
as well as the wild type were tested as pollutant sensors.

76
 Cont’d…
 To screen water samples for pollutants (metal or
organic) a suspension of P. fluorescens was mixed
with the solution to be tested.
 After a 15 min incubation the luminescence of the
suspension was measured.
 When the solution contained low to moderate levels
of pollutants the bioluminescence was inhibited.
 The procedure is rapid, simple, cheap and a good
screen for pollutants.

77
 Ba
ct
er
ia
l Bi
o se
78

ns
or
 2.2.2. Vaccine
Production
 A vaccine renders the recipient resistant
to infection.
 During vaccination a vaccine is injected
or given orally.
 The host produces antibodies for a
particular pathogen.
 Upon further exposure the pathogen is
inactivated by the antibodies and
disease state prevented.
 Generally to produce a vaccine the
pathogen is grown in culture and
inactivated or non-virulent forms are 79
 There are many disadvantages and they include:

 Not all organisms can be cultured.

 The procedure is expensive and
sometimes unsafe.
 New pathogens keep occurring.

 For some pathogens e.g. HIV

vaccination is not appropriate.
 Why?
80
  New Generation of
Vaccines
 Recombinant DNA technology is being used to
produce a new generation of vaccines.
 Virulence genes are deleted and organism is still
able to stimulate an immune response.
 Live nonpathogenic strains can carry antigenic
determinants from pathogenic strains.
 If the agent cannot be maintained in culture, genes of
proteins for antigenic determinants can be cloned
and expressed in an alternative host e.g. E. coli.

81
 There are three types of vaccines:

1. Subunit (protein) vaccines

2. Attenuated vaccines
3. Vector vaccines
1) Subunit Vaccines
 Antibodies usually bind to surface proteins of the
pathogen or proteins generated after the disruption of the
pathogen.
 Binding of antibodies to these proteins will stimulate an
immune response.
 Therefore proteins can be use to stimulate an immune
response.
82
 It has been showed that the capsid or envelope
proteins are enough to illicit an immune response.
Examples:-

 Herpes simplex virus envelop

glycoprotein O.
 Foot and mouth disease virus capsid
protein (VP1)
 Extracellular proteins produced by
Mycobacterium tuberculosis.
83
 A Subunit Vaccine for Mycobacterium
tuberculosis
 Tuberculosis is caused by Mycobacterium tuberculosis.
 The bacterium form lesions in the tissues and organs
causing cell death.
 Often the lung is affected.
 About 2 billion people are infected and there are 3
million deaths/year.
 Currently tuberculosis is controlled by a vaccine called
BCG (Bacillus Calmette-Guerin) which is a strain of M.
bovis.
 M. bovis often responds to diagnostic test for M.
tuberculosis.

84
 Six extracellular proteins of M. tuberculosis were
purified.
 Separately and in combinations these proteins were
used to immunized guinea pigs.
 These animals were then challenged with M.
tuberculosis.
 After 9-10 weeks examination showed that some
combinations of the purified proteins provided the
same level of protection as the BCG vaccine.
85
2. Attenuated Vaccines
 Attenuated vaccines often consists of a pathogenic
strains in which the virulent genes are deleted or
modified.
a)The Development of a Live Cholera Vaccine

 Live vaccines are more effective than a killed or

subunit (protein) vaccines.
 With this in mind a live vaccine for cholera was
developed.
 Cholera is characterized by fever, dehydration,
abdominal pain and diarrhea.
86
 The causal agent of cholera is Vibrio cholerae and
is transmitted through contaminated water.
 V. cholerae produces an enterotoxin with an A
subunit and 5 B subunits.
 Presently the cholera vaccine consist of a phenol-
killed V. cholerae and it only last 3-6 months.
 A live vaccine would be more effective.

 In the sequence of the A peptide a tetracycline

resistance gene is inserted.

87
 A plasmid with A peptide was digested with 2 restriction
enzymes Cla1 and Xba1.
 This removes 550 bases of A peptide.
 A Xba1 linker was added and T4 ligase used to ligate the
DNA. This plasmid was mixed with V. cholerae with
tetracycline resistant gene.
 By conjugation the plasmid was transferred to the strain
with the tetR gene inserted into it’s chromosomal DNA.
 By recombination the A peptide with the tetR gene was
replaced by the deleted A peptide.
 The final result is V. cholerae with a 550 bp of the A
peptide deleted. It can be used as a vaccine.

88
Production of
a Live Cholera
Vaccine

89
Production of
a Live Cholera
Vaccine

90
 3. Vector Vaccine
 A vector vaccine is a vaccine which is introduced
by a vector. e.g. Vaccinia virus
 The vaccinia virus as a live vaccine led to the
globally eradication of the Smallpox virus.
 The genome of the vaccinia virus has been
completely sequenced.
 The virus replicates in the cytoplasm rather than
in the nucleus.
 The vaccinia virus is generally non-pathogenic.
91
 These characteristics makes the
vaccinia virus a good candidate for a
virus vector to carry gene for
antigenic determinants from other
pathogens.

92
 A number of antigen genes have been inserted
into the vaccinia virus genome.
Examples:-
 Rabies virus G protein
 Hepatitis B surface antigen

 Influenza virus NP and HA proteins

 A recombinant vaccinia virus vaccine for rabies
is able to elicit neutralizing antibodies in foxes
which is a major carrier of the disease.

93
 2.2.3. Gene
 Human beingsTherapy
suffer from more than 5000 different genetic
diseases.
E.g. Cystic fibrosis, sickle cell anemia, hemophilia, etc.
In addition many common disorders like cancer, hypertension,
mental illness… etc have genetic components.
Gene therapy is the introduction of a normal functional gene
into cells, which contains the defective allele of concerned
gene with the objective of correcting a genetic disorder.
 Types of gene therapy:
a) Germline gene therapy
b) Somatic cell gene therapy

94
95
 A) Germline Gene Therapy
Germ cells i.e. sperms and eggs are modified by introduction of
functional genes, which are ordinarily integrated into their
genomes.
A fertilized egg is provided with a copy of the correct version of
the relevant gene and re-implanted into the mother.
If successful, the gene is present and expressed in all cells of the
resulting individual.
Therefore the change due to therapy would be heritable and would
be passed on to later generations.
Currently it is not yet applied for human beings due to lack of
development of the techniques and ethical issues.
It is currently applied for curing diseases in animals (lower high
blood pressure in rats) 96
B) Somatic Gene Therapy
 Somatic cell therapy involves manipulation of
somatic cells.
 Expression of the introduced gene eliminates
symptoms of the disorder, but this effect is not
heritable.
 Somatic cell therapy has potential in the treatment
of:
 Hemophilia
 Cystic fibrosis 97
98
 Mode of Delivery of the Functional Gene
A. Virus Mediated
 Employs recombinant retroviruses or adenoviruses
that carry the functional gene and express the
functional or correct gene in the host cell
 The major problem is that most viral DNA do not
integrate in to the host genome.
 However, adeno-associated virus has the ability to
integrate to chromosome number 19.
B. Non Viral Methods
 Naked DNA insertion
 Liposomes
 Electroporation
99
 2.2.4. Organ/Tissue
Transplantation
Definition:
 Transplantation- is the act of transferring an organ,
tissue, or cell from one place to another.
 The field of organ transplantation has made remarkable
progress in a short period of time.
 Transplantation has evolved to become the treatment of
choice for end-stage organ failure resulting from
almost any of a wide variety of causes.
 Transplantation of the kidney, liver, pancreas,
intestine, heart, and lungs has now become
commonplace in all parts of the world.
100
 Organ transplantation- is an effective treatment for
many patients facing severe debility or premature death
due to organ failure.
 Organ- A part of the body that performs vital
function(s) to maintain life (e.g. heart, lung, liver, kidney
and pancreas transplants are well-established procedures).
 Tissue transplantation- continues to develop rapidly,
and transplants of eye tissue, heart valves, skin and
musculoskeletal tissue are effective and well-established
therapies.
 Tissue- group of specialized cells that perform defined
functions.
 Rates of transplantation are limited by the availability of
organs and tissues.

101
  Type of Transplants
 Broadly speaking, transplants are divided into three
categories based on the similarity between the donor
and the recipient:
1. Autotransplants
2. Allotransplants
3. Xenotransplants
1. Autotransplants- involve the transfer of tissue or
organs from one part of an individual to another part
of the same individual.
 They are the most common type of transplants and
include skin grafts and vein grafts for bypasses.
 NO immunosuppression is required
102
2. Allotransplants- involve transfer from one
individual to a different individual of the same
species the most common scenario for most solid
organ transplants performed today.
 Immunosuppression is required for allograft
recipients to prevent rejection.
3. Xenotransplants- involve transfer across species
barriers. Currently, xenotransplants are largely
relegated to the laboratory, given the complex,
potent immunologic barriers to success.

103
104
105
106
 Transplant Immunobiology
 The success of transplants today is due in large part
to control of the rejection process, thanks to an
ever-deepening understanding of the immune
process triggered by a transplant.
Transplant Antigens
 The main antigens involved in triggering rejection
are coded for by a group of genes known as the
major histocompatibility complex (MHC).
 In humans, the MHC complex is known as the human
leukocyte antigen (HLA) system. It comprises a
series of genes located on chromosome 6.

107
 HLA molecules can initiate rejection and graft
damage, via humoral or cellular mechanisms.
Humoral Rejection- mediated by recepient's AB.
(e.g. blood transfusion, previous transplant, or
pregnancy)
Cellular Rejection- is the more common type of
rejection after organ transplants. Mediated by T
lymphocytes, it results from their activation and
proliferation after exposure to donor MHC
molecules.
108
 Complications of Organ Transplantation
1. Rejection
2. Malignancy
1. Clinical Rejection
 Graft rejection is a complex process involving several
components, including T lymphocytes, B lymphocytes,
macrophages, and cytokines, with resultant local
inflammatory injury and graft damage.
 Rejection can be classified into the following types based on
timing and pathogenesis:
 Hyperacute
 Acute and
 Chronic

109
A. Hyperacute Rejection
 This type of rejection, which usually occurs within
min after the transplanted organ is reperfused, is
because of the presence of preformed antibodies
in the recipient, antibodies that are specific to
the donor.
 These bind to the vascular endothelium in the
graft and activate the complement cascade,
leading to platelet activation and to diffuse
intravascular coagulation.
 The result is a swollen, darkened graft, which
undergoes ischemic necrosis. 110
B. Acute Rejection
 This used to be the most common type of
rejection, but with modern immunosuppression it
is becoming less and less common.
 Acute rejection is usually seen within days to a
few months post-transplant.
 It is predominantly a cell-mediated process,
with lymphocytes being the main cells involved.
 With current immunosuppressive drugs, most
acute rejection episodes are generally
asymptomatic.
111
C. Chronic Rejection
 This form of rejection occurs months to years post-
transplant.
 Now that shortterm graft survival rates have
improved so markedly, chronic rejection is an
increasingly common problem.
 Histologically, the process is characterized by
atrophy, fibrosis, and arteriosclerosis.
 Both immune and nonimmune mechanisms are
likely involved.
 Clinically, graft function slowly deteriorates over
months to years
112
 Clinical Immunosuppression
 The success of modern transplantation is in large
part because of the successful development of
effective immunosuppressive agents.
 Two types of immunosuppression are used in
transplantation:
 Induction and
 Maintenance immunosuppresion
1. Induction Immunosuppression
 refers to the drugs administered
immediately posttransplant to induce
113
2. Maintenance Immunosuppression
 refers to the drugs administered to maintain
immunosuppression once recipients have recovered
from the operative procedure.
 Individual drugs can be categorized as either
biological or non-biological agents.
 Biological agents (monoclonal and polyclonal
antibodies) consist of antibody preparations directed
at various cells or receptors involved in the rejection
process; they are generally used in induction (rather
than maintenance) protocols.
 Non-biological agents (e.g. corticosteroids,
azathioprine and cyclosporines)form the mainstay of
maintenance protocols.
114
 2. Malignancy
 Transplant recipients are at increased risk for developing
certain types of de novo malignancies, including non-
melanomatous skin cancers (3–7-fold increased risk),
lymphoproliferative disease (2–3-fold increased risk),
gynecologic and urologic cancers, and Kaposi
sarcoma.
 The risk ranges from 1 percent among renal allograft
recipients to approximately 5–6 percent among recipients
of small bowel and multivisceral transplants.
 The most common malignancies in transplant recipients
are skin cancers.
115
 2.2.4. Forensic Medicine
 It is a branch of medicine that provides evidences for
legal cases such as determination of cause of death,
identification of criminals etc.
 DNA fingerprinting is one of the methods employed in
forensic medicine.
 It is the characterization of relatively one or more
relatively rare features of an individuals genome or
hereditary make up.
 DNA Fingerprinting for Identification of Crime
Suspects
 It is almost impossible for a person to commit a crime
without leaving behind a trace of his or her DNA. E.g.
Hairs, spots of blood.
116
 The human genome is more or less the same in
everybody the same genes will be in the same
order.
 But the human genome, as well as other organisms,
show many polymorphisms
 Polymorphisms refers to nucleotide sequence that
are not found in the same positions and or in the
same order of sequence.
 The 1st DNA fingerprinting is based on a kind of
variation in the human genome called a hyper
variable dispersed repetitive sequence.

117
As the name indicates, this is a repeated sequence that
occurs at various places (“dispersed”) in the human
genome.
The key feature of these sequences is that they are located
at different positions in the genomes of different people

118
 Preparation of DNA Fingerprints
 a sample of DNA is digested with a restriction
endonuclease,
 the fragments are separated by agarose gel
electrophoresis,
 a Southern blot is prepared
 The fragments are hybridized with a labeled probe
complementary to the repeat sequence
 The label signals a series of bands, each one
representing a restriction fragment that contains the
repeat.
 Because the insertion sites of the repeat sequence are
variable, the same procedure carried out with a DNA
sample from a second person will give a different
pattern of bands.
 These are the genetic fingerprints for those individuals.
119
After restriction digestion

120
 In the case of solving crimes, DNA samples from the
crime scene and possible suspects are collected and
DNA fingerprints are generated
 If there is a match between the DNA fingerprints of a
suspect and the DNA fingerprint from the crime scene
then, the suspect is the criminal.

DNA fingerprint of DNA DNA fingerprints of suspects

sample from the crime 1&2
scene

121
2.2.5. Stem cell Research and Its Potential in
Medicine
 Stem Cell- is  type of cell that can make any kind of cell
required to build an organism.
 When a stem cell divides, one new cell that results can
remain a stem cell while the other new cell becomes an
ordinary cell with a particular function in the organism.
 Sometimes a stem cell that divides produces two identical
stem cells.
 Stem cells can renew themselves indefinitely.
 The ability of stem cells to produce new cells of specific
types is of special interest to medical science.
 Medical researchers and other scientists study stem cells to
understand basic processes in cell development and disease.

122
 The special properties of stem cells make them potentially
powerful medical tools that could repair or replace
diseased or injured tissues or organs in humans.
 Research is usually done with stem cells from mice or
from humans.
 Types of Stem Cells
 Two basic types of stem cells occur in humans and
animals:
 Embryonic stem cells and
 Adult stem cells
 Embryonic stem cells have the potential to develop into
any organ or type of tissue in the body.
 This property is called pluripotency.
123
 Adult stem cells also called somatic stem cells, are found in humans
and animals after birth and remain active in the body throughout a lifetime .
 Adult stem cells can only turn into certain specialized
types of cells.
 Different types of adult stem cells act as a repair system
and are able to replace such cells as blood cells, bone
cells, or certain nerve cells in the body.
 Researchers continue to discover new types of adult
stem cells in different parts of the body and have
discovered that blood stem cells originate in the
placenta.
 In living humans and animals, development of adult
stem cells appears to be influenced by their niche or
microenvironment in the body, which restricts what they
become.
124
 Cancer stem cells are found in some tumors and in some
blood cancers ( leukemia), and appear to be a factor in
making such cancers grow.
 Similar to normal adult stem cells, cancer stem cells can
divide to produce a new cancer stem cell and a particular
type of cancer cell.
 Growing Stem Cells from which all human tissues develop,
may provide powerful tools in the treatment of disease.
 To explore their potential uses, scientists can theoretically
grow stem cells from leftover eggs fertilized by sperm
during laboratory fertility treatments.
 After several days, the fertilized egg forms a mass called a
blastocyst with stem cells inside.
 The cells are removed from the blastocyst and grown in
laboratory dishes into specialized body cells
 Scientists have reported success in growing nerve, bone,
muscle, blood, and skin cells.
125
126
 To explore potential medical uses of stem cells, scientists need to
produce stem cell lines.
 These lines are colonies of stem cells that grow and replicate
themselves in culture that is, in a special nutrient substance in a
laboratory dish.
 A stem cell line provides a scientist with a virtually endless
supply of material to explore and test.
 Stem cells; however, seem to lose some of their ability to make a
wide range of cells as they age.
 In other words, an older stem cell may not be as versatile as a
younger one.
 The exception seems to be adult stem cells derived from bone
marrow, which retain their ability to transform into any cell type.
 As a result, adult stem cells from bone marrow and the earliest
stem cells those found in an embryo may provide the most
powerful tools for use in medical treatment.
127
 2.2.6. Protein Based Drugs
 Before the advent of molecular biotechnology most
human proteins were available in only small
(limited) quantities.
 Today hundreds of genes for human proteins
have been cloned, sequenced, expressed in the
host cells and are being tested as therapeutic
agents (drugs) in humans.

128
a) Enzymes as Therapeutic Agents/ DNase1
 Cystic fibrosis (CF) is one of the most fatal heredity
diseases among European and their descendants with
~30,000 cases in the US and 23,000 in Canada.
 Furthermore among European descendants it occurs in
1 in 2,500 live birth and 1 in 25 are carriers.
 It is caused by more than 500 different mutations in
the cystic fibrosis trans membrane conductance
regulator (CFTR) gene.
 Individuals with CF are highly susceptible to bacterial
infection and antibiotic treatment often results in
resistant strains.

129
 DNase 1
A thick mucus which is a results of:
 Alginate produced by bacteria
 DNA from lysed cells
 Leucocytes which accumulate due to the
infection
 Makes breathing difficult.
 Scientist at Genentech isolated the gene for DNase1
 The purified enzyme was delivered as an aerosol to the
lung where it hydrolyzed the DNA into short oligonucleotides.
 This decrease the viscosity in the lungs and made breathing
easier.

130
 Alginate Lyase
 Alginate is a polysaccharide polymer that is produced by
seaweed and some soil and marine bacteria.
 The excretion of alginate by Pseudomonas aeruginosa of
patients with CF contributes to the viscosity in the lung.
 The enzyme alginate lyase can liquefy bacteria alginate.
 Alginate lyase was isolate from Flavobacterium sp. and
cloned into E. coli.
 The expressed gene produced a protein of 69,000 Da.
 The 69,000 Da protein produced a proteolytic enzyme of
6,000 Da.
 The remain 63,000 Da protein was cleaved to produce a
43,000 Da which is able to liquefy bacterial alginate.
 Combined with NDase1, alginate lyse is able to reduce the
mucus in the lungs of patients with CF. 131
 DNase 1 and
Alginate lyase

132
The end!

133
 Chapter 3
Agricultural Biotechnology
3.1. Plant Cell and Tissue Culture
Definition and History
 Tissue culture broadly refers to:
‘‘ the technique of growing plant cells, tissues, organs,
seeds or other plant parts in a sterile environment on a
nutrient medium.’’
‘‘ aseptic culture of plant protoplast, cells, tissues and
organs under conditions which lead to cell multiplication
and regeneration of organs or whole plants’’
 The culture system is based on the totipotent
characteristics of plant cells which is the ability to change in
to meristematic state and differentiate in to a whole plant.

134
 Some of the Landmark Discoveries in
Plant Tissue Culture
 1902- Haberlandt (father of plant tissue culture)
proposed the concept of in vitro culture & was able to
culture differentiated plant cells; but did not regenerate
whole plant.
 1921- Milliard had limited success in cultivation of
plant embryos.
 1934- White cultured tomato root cells for a long period
of time.
 1939- Gautheret, White and Nobecourt were successful
in regenerating whole plant.
135
 1960- Cocking produced large amounts of
protoplasts using cellulase enzymes
 1962- Murashige and Skoog developed the most
commonly used medium MS medium.
 1964- Maheshwari and Guha regenerated the
first haploid plants (Datura) from pollen culture
 1972- Carlson produced first interspecific somatic
hybrid of Nicotiana species by protoplast
fusion.

136
3.1.1. Conditions for Plant Tissue Culture
 The techniques in plant tissue culture must set conditions that:
 keep plant cells and organs free from microbes
(bacteria & fungi)
 ensure desired developments in the cells and
organs by providing suitable nutrient media and
other environmental conditions.
 Organization of the Tissue Culture Laboratory
 It depends on the scale of activity. But generally, the laboratory
should have space for:
I. Washing, drying and storage facility
II. Preparation, sterilization and storage of media
III. Aseptic handling of explants and cultures
IV. Maintenance of culture
137
I. Washing, Drying and Storage Facility
 For washing, an area with large sinks and draining
system is necessary.
II. Preparation, Sterilization and Storage of Media
 Includes the area for media preparation and
sterilization.
 Should have ample storage and bench space for:
 Chemicals
 Glass wares
 Culture vessels and
 Other items needed for media preparation.

138
 Some of the Items Required for
Tissue Culture Laboratory are:
 Laminar airflow hood
 Autoclave or pressure cookers
 Refrigerators
 Electronic balances
 pH meter
 Water distillation unit
 Magnetic stirrer with hot plate
 Water bath with thermostat
 Ovens with thermostat
 Vortex
 Centrifuges
 Pipettes
 Different glass wares and etc.
139
III. Transfer Area for Aseptic Handling of
Explants and Cultures
 Sterile, dust free room should be available for routine
transfer and manipulation work
 Laminar airflow cabinet is the most common
accessory used for aseptic manipulation.
 In airflow cabinet, air is forced into a cabinet through a
bacterial HEPA (High Efficiency Particulate Air) filter.
 This prevents bacteria from entering to the hood and
settling on the bench.
 The roof of the cabinet houses an ultraviolet (UV) light
used to sterilize the interior of the chamber.
 The UV light is turned on 30 min prior to using the
chamber but must be turned off during operation.
 It is also necessary to disinfect the inside of the cabinet,
especially the table top with 70 % alcohol.
140
141
IV. Maintenance of Culture and
Planting
Regenerated Plants
 Plant tissue cultures should be incubated in a room under
conditions of well controlled temperature, illumination,
photoperiod, humidity and air circulation.
 In addition, a dark area is necessary if some cultures require
continuous darkness.
 Green houses are required to grow plantlets for further
propagation and for growing plants to maturity.
 This facility is required as a transitional step from culture
containers in the controlled room to the field.
 In the green house plants are acclimatized and hardened
(develop adequate root systems and leaf structures to
withstand the field environment) before being transferred to
the field conditions.
142
143
 3.1.2. Medium Composition
 An explant is a cell, tissue or organ, excised from a
plant, used to start in vitro cultures.
 When the explant is excised, it is no longer able to
receive nutrients or hormones from the mother
plant.
 These components must be provided in vitro to allow
growth.
 The composition of the artificial nutrient medium may
vary for different species and purpose of culture.
 A nutrient medium is defined by its mineral salt
composition, carbon source, organic supplements and
plant growth regulators.
144
145
146
 Mineral Salts / Inorganic Nutrients
 In vitro growth of plants requires combination of
macro- and micronutrients that is similar to the
soil composition.
 Macronutrients
 are classified as those elements which are required
in concentration > 0.5mM/L.
 include N, K, P, Ca, Mg and S in the form of salts.
 Micronutrients
 are those elements, which are required at a
concentration < 0.05mM/L.
 include Fe, Mn, Zn, B, Cu, Mo, Co, I, Ni, Al, Si.
147
148
149
 Different scientists have developed different
composition of
macro and micronutrients for culturing different
plants.

150
  Carbon Sources
 Carbon is required as energy source, since most plant
cultures are unable to photosynthesize effectively due to:
 inadequately developed cellular and tissue
development
 lack of chlorophyll
 limited gas exchange and carbon dioxide in tissue
culture vessels etc.
 Hence they lack autotrophic ability and need external
carbon for energy.
 The most preferred carbon or energy source is sucrose at a
concentration of 20-60 g/l.
 While autoclaving the medium, sucrose is hydrolysed to
glucose and fructose, which are then used up for growth.
151
  Organic Supplements
A. Vitamins:
 are required for metabolic processes as cofactors or
parts of enzymes.
 Thiamine (B1), nicotinic acid (B3), pyridoxine
(B6), pantothenic acid (B5) are commonly used
vitamins
B. Amino acids:
 Addition of amino acids to media is important for
stimulating cell growth
 This reduced organic nitrogen is more readily taken
up by plants than the inorganic nitrogen.
 L-glutamine, L-asparagine, L-cysteine, L-
glycine are commonly used amino acids 152
C. Complex Organic Molecules:
 These are groups of undefined supplements such as casein
hydrolysate, coconut milk, yeast extract, orange juice,
tomato juice, etc.
 These compounds are often used when no other combination
of known defined components bring the desired growth.
D. Activated Charcoal:
 acts both in promotion and inhibition of culture growth
depending upon plant species being cultured.
 It is reported to stimulate growth and differentiation in
orchids, carrot and tomato whereas inhibits tobacco,
soybean, etc.
 The other function is to absorb brown/ black pigments and
oxidized phenolics produced during culture and thus reduce
toxicity.
153
 Plant Growth Regulators (PGRs)
 PGRs stimulate cell division and hence regulate the
growth and differentiation of shoot and roots on explants
and embryos in semisolid or liquid medium cultures.
 The four major PGRs used are auxins, cytokinin,
gibberellins and abscissic acid.
A. Auxins
 Induce:
 cell division
 cell elongation
 apical dominance
 adventitious root formation and
 somatic embryogenesis, but
 inhibit adventitious shoot formation
154
 When used in :
 low concentration, auxins induce root initiation and
 high concentration, callus formation occurs.
 Commonly used synthetic auxins are:
 1-naphthaleneacetic acid (NAA)
 2,4 dichlorophenoxyacetic acid (2,4-D)
 indole-3 acetic acid (IAA)
 indolebutyric acid (IBA)
B. Cytokinins
 also promote cell division
 mainly stimulate initiation and growth of shoots.
 modify apical dominance by promoting axillary shoot
formation.
E.g. Zeatin, 6- benzylaminopurine (BAP) & kinetin
 When used in high concentration, it inhibits root
formation and induces adventitious shoot
formation. 155
C. Gibberellins , Abscissic acid and Ethylene
 are lesser used PGRs in plant tissue culture
 Gibberellic acid- is mostly used for internode
elongation; meristem growth; induces callus growth and
elongation of dwarf plantlets.
 It usually inhibits adventitious root and shoot
formation.
 Abscissic acid (ABA)- is used only for somatic
embryogenesis and for culturing woody species.
 It is a growth inhibitor, which seems to be synthesized
when a plant is under difficult condition.
 Ethylene- is a gaseous compound produced by
cultured cells, but its role in cell and organ culture is not
known.
156
  Solidifying Agent
 The tissue culture media should be semisolid to station
explants on the surface.
 As a solidifying agent, agar is added to the medium in
concentration ranging from 0.5 to 1 % (w/v).
 Agar is preferred over other gelling agents because it is
inert, does not react with media constituents and it is not
digested by plant enzymes.
 Agarose, a purified extract of agar is used for
protoplast culture.
pH
 the optimal pH for tissue culture is between 5.0- 6.0.
 Buffers are not required to maintain the pH in the media.
 The pH is usually adjusted using stock solutions of HCl
and NaOH.
157
  Types of Cultures
A. Callus Culture
 Culture of explants (from any part of the plant) using certain
combinations of PGR gives rise to an unorganized, growing and
dividing mass of cells called callus.
 If actively dividing cells (meristematic cells, shoot tips, buds,
immature leaves etc) are used as explants, cell division and
multiplication will be rapid b/c immature tissues are more
morphogenetically flexible.
 During callus formation, there is some degree of
dedifferentiation both in morphology (callus is usually
composed of unspecialized parenchyma cells) and
metabolism.
 One major consequence of this dedifferentiation is that most
plant cultures lose the ability to photosynthesize.
 Callus culture is often performed in the dark as light can
encourage differentiation of the callus. 158
 Callus cultures can be manipulated for different
purposes by changing the hormone
concentrations in the media.
 Application
 Callus cultures show slow growth. It can be used to
study plant growth, differentiations and metabolism.
 Callus culture is the intervening step for indirect
organogenesis.
B. Seed Culture: is the culture of seeds in vitro to
generate seedlings/plants.
 Seed culture increases the efficiency of germination
of seeds that are difficult to germinate in vivo.

159
C. Embryo Culture: is the culture of isolated mature or
immature embryos to obtain a viable plant.
 Embryo culture is used for:
 overcoming embryo abortion due to
incompatibility barriers from crosses between
unrelated plants.
 overcoming seed dormancy (especially trees)
e.g. Seeds of banana that will never germinate
under normal conditions can be cultured in vitro.
D. Organ Culture: is the culture of isolated plant organs, e.g.
meristem, shoot tip, root culture, anther tissue culture.
E. Cell culture: is the culture of isolated cells or very small
cell aggregates remaining dispersed in liquid medium.
F. Protoplast culture: is the culture of plant protoplasts.

160
 Plant Regeneration
 There are two methods of whole plant
regeneration:
 Organogenesis and
 Somatic embryogenesis
1. Organogenesis
 Organogenesis is the production of plant organs
(root, shoot etc) either directly from explants or
indirectly by initiating a callus followed by organ
differentiation.
Direct Organogenesis
 The somatic tissues of higher plants are capable,
under certain conditions, of regenerating
adventitious plant parts, which are not normally
produced by the organ. 161
 Manipulation of auxin to cytokinin ratio in the medium lead
to the development of shoots, roots from which whole
plants can subsequently be produced.
 Low concentration of auxin: cytokinin induces adventitious
shoot formation while
 High concentration of auxin: cytokinin induces root
formation
 Direct organogenesis is especially suitable for
herbaceous plants.
 The adventitious plant parts generated (mainly the shoots)
are frequently used as raw materials for micropropagation.
Indirect Organogenesis
 It involves an intervening callus formation stage before
organ differentiation.
 Similarly, varying the concentration of auxin and cytokinin
from the callus culture allows regeneration of the plant.

162
2.Somatic Embryogenesis
 It involves redifferentiation of meristematic cells into non
zygotic somatic embryo, which are capable of germinating to
form complete plants.
 Somatic embryogenesis differs from organogenesis in the
embryo, being a bipolar structure rather than monopolar.
 Furthermore, induction of somatic embryogenesis requires
a single hormonal signal while in the organogenesis two
different hormonal signals are needed to induce first a
shoot organ, then a root organ.
 The presence of auxin (generally 2,4-D) in the medium is
essential for induction phase.
163
 Embryogenesis can also be either direct or indirect.

Direct Embryogenesis
 The embryo is initiated directly from the explants that
have pre-embryogenic determined cells.
 Such cells are found in embryonic tissues (e.g.
scutellum of cereals), hypocotyls and nucellus.

Indirect Somatic Embryogenesis

 Callus is first produced from the explants.
 Embryos can then be produced from the callus tissue
or from a cell suspension produced from that callus.

164
 Somatic embryogenesis involves three distinct
steps which are absent in organogenesis:
 Induction: cells of callus are induced to divide and
differentiate into groups of meristematic cells
called embryogenic clumps (ECs).
 These clumps break the cytoplasmic connections
and fuse to develop into initial stages of somatic
embryo i.e. globular stage (round bell shape).
 Maturation: somatic embryos develop into
mature embryos by differentiating from globular
to heart to torpedo to cotyledonary stages.
 The mature embryo here undergoes biochemical
changes to acquire hardiness.
 Conversion: Embryos germinate to produce
seedlings.
165
166
3.1.3. Micropropagation
It is the in vitro vegetative propagation of plants by tissue culture.
The chief objective of micropropagation is to produce large numbers
of progeny plants, genetically identical to the parents.

Stages of Micropropagation
Stage 1. Preparation of Parent Plant
It involves the selection and growth of a healthy stock plant under
controlled conditions (in a green house).
Step 2. Preparation of Explants
Healthy leaves, stems, or other suitable plant parts are selected and
cut from the mother plant.
Dust particles are removed from the tissue by washing with water.
The explant is immersed in disinfectants (alcohol/sodium
hypochlorite/ mercury chloride/ H202) for surface sterilization.
Excess chemical from the surface is washed off using sterile water.
167
 Step 3. Initiation of Culture and Multiplication of
Shoots
 The surface sterilized explant is implanted into a
suitable nutrient medium.
 The nutrient media composition should have the correct
type of growth hormones in appropriate concentrations for
organogenesis or somatic embryogenesis.
 Plant regeneration can achieved either through direct or
indirect organogenesis or somatic embryogenesis
depending on the explant and plant species used.
 For instance, if indirect organogenesis is followed, callus
is initiated and transferred to a medium having the
hormone combination favoring shoot formation.

168
 Nodes and/or shoots are excised from this culture and
transferred to a fresh media to generate more shoots.
 Then, these shoots are transferred to another
medium, which favors the root initiation.
Step 4. Transplantation and Hardening
 The plantlets that develop roots can be transferred to
special pots with sterilized sand for hardening under
greenhouse conditions.
 Hardening is a process by which the in vitro generated
plants are acclimatized to the greenhouse
conditions.
 Then, they can be transferred to the field.
169
 The advantages of plant tissue culture over
conventional vegetative propagation include:
 It is rapid and utilizes small space to produce large number
of plantlets.
 The plant cultures require less attention and labour cost.
 Micropropagation can be carried out throughout the year
independent of the seasons.
 Since asceptic conditions are followed, the plants
propagated are free from diseases (fungal and bacterial).
 It requires only small explants. Therefore it is valuable for
propagating plants that have limited tissue available as
explants.
 It is suitable for rapid propagation of forest trees, which
usually produce dormant seeds.
 In the case of ornamental plants, tissue culture allows
better growth and flowering and may also acquire desirable
characteristics (short and bushy plants).
170
3.1.4.Cell Suspension Cultures and Secondary
Metabolites
 Culturing tissues and cells in a liquid nutrient
medium produces a suspension of single cells and
cell clumps called suspension culture.
 Single-cell cultures and suspension cultures can be
established from callus cultures by transferring a
piece of callus tissue into liquid medium and
subjecting it to continuous shaking
 The growth rate of the suspension-cultured cells
is generally higher than that of the solid culture
and hence need to be subcultured more frequently.

171
172
 Cell suspension cultures are important for
production of secondary metabolites from plants.
 Secondary metabolites are chemicals that are formed
either
 to attract pollinators,
 to combat infectious diseases or
 to warn off predators.
 If plant’s cells are cultured to attain a certain degree
of differentiation, then it is possible to obtain
secondary metabolites produced by specific cells/
tissues.
 Examples: alkaloids, flavanoids, tannins, terpins,
essential oils, latex, etc.
 These chemicals provide industrially important
natural products like color, insecticides,
therapeutic drug components, fragrances, etc
173
 Examples of secondary metabolites produced from cell
suspension cultures

174
 There are two types of suspension cultures:
 Batch and
 Continuous cultures.

A.Batch Culture
 Cells are inoculated into a fixed volume of medium.
 They consume nutrients as they grow and release toxic
metabolites into the surrounding medium.
 This results in the continuous variation in the chemical
and physical environments such as osmolarity, pH, and
depletion of nutrients.
 All these variations eventually cause cells to cease
multiplication and die.
 These cultures can be maintained continuously by sub-
culturing i.e. by transferring a small aliquot of inoculum
from the grown culture to fresh medium at regular
intervals.
175
 The biomass or cell number of a batch culture
follows a typical sigmoidal curve with 5 phases:
I. Lag Phase
 Preparatory phase where cells prepare to divide.
 The cell number is constant
II. Exponential or Log phase
 There is a rapid increase in cell number because the rate
of cell division is highest.
III. Linear Phase
 Cell division slows but rate of cell expansion increase
IV. Stationary Phase
 The number and size of cells remain constant
V. Deceleration Phase
 rates of cell division and elongation decreases.
 The cells stop dividing due to depletion of nutrients
and accumulation of cellular wastes. 176
 Plant Cell Suspension typical
Growth curve

16
14
Dry weight (g/l)

12
10
8
6
4
2
0
0 2 4 6 8 10 12 14 16 18 20 22
time (d)

177
B. Continuous Culture
Steady state of cell density is maintained by regularly replacing
a portion of the used up medium with fresh one.
Continuous culture is further classified into two types

Closed Type
The used medium is replaced with fresh medium,

But the cells from the used medium are mechanically retrieved
and added back to the culture and
Thus, the cell biomass keeps increasing.

Open Type
Both cells and used medium are replaced with fresh medium thus
maintaining culture at constant and sub-maximal growth rate.
178
3.1.5. Somaclonal Variation and Somatic
Hybridization
 Somaclonal Variation
 Under certain conditions, somatic cells from plant tissue
culture show genetic variability.
 This is termed as somaclonal variation.
 This variation results in the formation of
 Altered ploidy
 Sterile plants and
 Morphological variants
 Some of which may involve traits of economic importance
for crop plants.
 Crop improvement was observed due to somaclonal variation.
 The first observations of improvements were somaclones of
 sugarcane resistant to downy mildew disease &
 potato resistant to early blight.
179
 Some of the Major Causes of Somaclonal
Variations Include:
I. Culture Media Composition
 It has been assumed that certain constituents of the culture
medium, particularly certain growth regulators (2,4-D;
2,4,5-T) are mutagenic.
II. Growth Pattern and Mode of Regeneration
 There are suggestions that regeneration via
embryogenesis gives better chances of obtaining
genetically uniform plants than through organogenic
differentiation.
III. Length of Culture Period and Frequency of
Subculture
 Karyotypes show variation with increasing age of callus.
Generally, the proportion of variant plants produced during
successive passages also increases.
 It has been suggested that frequent transfers compared to
extended subculture intervals, yield more stable cultures.
180
 IV. Ploidy and Genotype
 Ploidy level and genotype of the plant determine the
susceptibility of cells to in vitro changes.
V. Physical Factors
 Physical state of the medium also influences the cytological
behavior of the cultured cells.
 In some explants, ploidy levels
 Increases when cells were cultured in suspensions but
 decreases when they were re-cultured as callus on a
solid medium.
 Culture temperature can influence the rate of mutation.
Tobacco callus incubated at
 35oC remained predominantly diploid,
 at 25oC showed marked karyological instability and
became mainly tetraploid.
181
  Somatic Hybridization
 Protoplast from two different plants can be fused to create a
hybrid.
 This technique of hybrid production through the fusion of body
cells, bypassing sex altogether, is called somatic
hybridization.
 Unlike sexual reproduction in which organelle genomes are
generally contributed by the maternal parent, somatic
hybridization also combines cytoplasmic organelles from
both parents.
 Fusion products with the nucleus of only one parent and
extra-nuclear(organelle) genome/s of the other parent are
referred to as cybrid and the process to obtain cells or plants
with such genetic combination/s is called cybridization. The
nucleus of the other parent is irradiated and is therefore not
viable.
 Protoplasts can be induced to fuse by chemical or electrical
manipulations which induce membrane instability. 182
 Applications of Somatic Hybridization
Genetic recombination in asexual or sterile plants:
 Genomes of asexually reproducing plants can be
recombined using this approach to produce hybrids of
practical breeding value.
Genetic recombination between sexually
incompatible Spp:
 The incompatibility barriers in sexual recombination at
interspecific or intergeneric levels are also overcome by
somatic hybridization.
 Generally, somatic hybrids are used for transfer of useful
genes such as disease resistance, abiotic stress resistance
or genes of industrial use.

183
 Cytoplasm Transfer
 This method allows cytoplasm transfer between
sexually incompatible species.
 This approach has been potentially used to
transfer two desirable traits – cytoplasmic male
sterility (CMS) and resistance to atrazine
herbicide, both coded by cytoplasmic genes.

184
Overall, plant tissue culture technique is
applicable for:
 regeneration of genetically engineered cells;
 fast commercial propagation of new plant
(micropropagation);
 cloning of rare and endangered plants;
 studying plant genetics, physiology and morphology;
 studying plant diseases and producing disease free
plantlets;
 production of variant clones with new characteristics
(somaclonal variation);
 production of haploids for improving crops (haploid
culture) &
 production of pharmaceuticals (cell suspension).
185
 3.2. Gene Transfer in Plants
 Improvement in food crop has been and is currently
achieved through conventional breeding techniques.
 However, it takes several years (6- 11) to develop a new
plant variety.
Genetic engineering allows much more precise
manipulation of plant genes in a short period.
Plant genetic engineering deals with the transfer of
genes of interest sourced from another plant or other
organisms and expression in to a host plant cell.
Introduction of exogenous DNA into eukaryotic cell is
called Transfection. However, molecular biologists do
not use this term and use transformation for all
organisms.
The transferred DNA is referred to as transgene;
 the process of transformation is called plant transgenesis and
 the transformed plants are called transgenic plants.
186
 The end product of transformation is not a transgenic cell
but a transgenic whole plant.
 Therefore, a transgenic plant is developed by integrating:
 rDNA technology and
 tissue culture techniques.
 Plant cells possess a unique characteristic called totipotency.
 It is the ability of a single plant cell to develop and
regenerate in to a whole plant.
 Any specialized plant cell can reverse the process of
differentiation and give rise to any type of cell.
 Regeneration of the whole plant from a single cell can be
achieved on artificial medium using tissue culture
techniques.
 Therefore, a transformed plant cells, carrying the DNA
insert gives rise to a transgenic plant which carries the
cloned DNA on to its progeny following flowering and seed
formation.
187
188
 Methods of Gene Transfer
I. Direct Methods
 Foreign DNA is inserted in to plant cell without the use of
vectors.
 These methods are simple and effective.
 They can be classified as physical and chemical gene
transfer method.
Physical Methods
 The methods include:

A.Electroporation
 It is applicable for transformation of intact plant cells and
protoplasts.
 Protoplast is a naked functional plant cell without the cell
wall.
 There are different methods of isolating protoplasts.
189
 Mechanical Method
 Select epidermis from plant cell
 Subject the cells to plasmolysis by suspending in a salt
solution.
 The protoplast shrinks away from the cell wall
 Dissect the tissue to release the protoplasts.
 Limitations of the method
 It is vey tedious
 Low yield of protoplasts
Enzymatic Method
 Utilizes enzymes that degrade the structural components
that make up the cell wall.
 The enzyme include:
 Pectinase to degrade pectin of the middle
lamella, resulting in disaggregation of cells and
 Cellulase to remove cellulose from the primary
and secondary cell wall.
 The major limitation of transforming protoplast is that it is
difficult to regenerate whole plant from these cells. 190
B. Gene gun/ Particle Bombardment
 Is the most effective method
 Successful for cereals (rice, maize etc)
 It is advantageous in transformation of organized
tissues.
C. Microinjection
 The plant material may be an intact cell, protoplast,
callus (undifferentiated aggregate of plant cells),
embryo, meristems etc.
D. Liposome Fusion
 Liposomes are artificially created lipid vesicles
capsuled by a phospholipid membrane to carry DNA
molecules.
 Liposomes have the ability to fuse with protoplast
membranes and allow the transfer of DNA.
 Efficiency of transformation is enhanced when it is
treated with Polyethylene glycol (PEG).
 The method applies for transformation of only
protoplast cells. 191
192
 Chemical Methods
A. Polyethylene Glycol Mediated Transformation
 PEG is a negatively charged compound that
precipitates DNA on the surface of protoplasts and
induce endocytosis.
 In the CaCl2, PEG co-precipitates with Ca and
induce the plasma membrane to take up DNA.
 This technique allows the transformation of large
numbers of protoplast cells.
 However, there is a possibility of DNA degradation
and allow the transfer of scrambled and multiple
gene copies in to the plant cells.

193
B. Calcium-phosphate Co-precipitation
Mediated Transfer
 The DNA is mixed with calcium chloride and
phosphate buffer.
 This forms a DNA-Ca-phosphate precipitate.
 Exposure of the precipitate to actively dividing
194
cells cause transformation.
II. Indirect/ Vector Mediated Transformation
 Utilizes either bacterial or viral vector for inserting
the gene of interest in to the plant genome.

A. Agrobacterium Mediated Transformation

 Utilizes a vector sourced from bacteria:
 Agrobacterium tumefaciens
 Agrobacterium rhizogenes
Agrobacterium tumefaciens is a soil borne
bacterium that causes crown gall disease in plants.
The transferred DNA causes a tumor or Crown gall
disease to plants.

195
 The bacteria infects plants which have wounds.
 Genes involved in crown gall disease are not
present on the chromosome of A. tumefaciens
but on a large plasmid, called the Ti (tumor-
inducing) plasmid.
 Only a portion of the Ti plasmid is transferred from
bacterial cells to plant cells.
 This portion of Ti plasmid is called T-DNA (Tumor
DNA).
 T-DNA can integrates stably into plant genome.

196
197
 When the Agrobacterium is in contact with the
damaged plant tissue, the chemicals (phenols)
trigger vir genes on the plasmid.
 Expression products of the vir genes trigger
 the replication of a ss T- DNA region from the Ti
plasmid.
 cleavage of the T-DNA & export into the plant cell
with protection.
 Upon entry in to the plant cells, it gets integrated
in to the plant genome.
 The T-DNA carries genes that code for opines and
agropines (amino acid derivatives) and cytokinin
and auxin (plant growth regulators).

198
 When the T-DNA is integrated in the plant genome,

 the amino acid derivatives are

expressed and
 synthesize the amino acid (aa) that are
used as a source of carbon and energy.
 while the hormones cause the plant cells
to proliferate and cause tumor cells.
 This interferes with the normal growth of
the plant.
 Agrobacterium infects dicot plants. 199
200
 Transfer of T-DNA from Agrobacterium to
plant cell

Agrobacteri wound
‘signal’
Plant cell
um T-
DN
A

Nucleus

Single-stranded copy of T-DNA

(protected by ssDNA binding protein)

201
 Cont’d...

Agrobacterium wound
‘signal’
Plant
T-
DN
A
cell

Nucleus

T-DNA
pore

Single-stranded copy of T-DNA

(protected by ssDNA binding protein)

202
 Therefore, the bacterium is a natural genetic engineer
that transforms plant cells and creates a biosynthetic
machinery to produce nutrients for its own use.
 This ability can be exploited to produce transgenic
plants. i.e. Ti plasmid can be used to transport any
gene of interest into plant cells.
 All that would be necessary would be to insert the gene
of interest into the T-DNA and
 Then, the bacterium could do the hard work of
integrating them into the plant chromosomal DNA.

203
204
205
B. Plant Viruses as Cloning Vectors
 A virus infects plant by inserting its DNA in to the cell.
 The viral DNA acts as a vector and does not integrate
in to the plant genome.
 Since the infection is systemic, it is easily transmitted
from one cell to another.
 The viral DNA can serve as a vector if it carries a
transgene.
 Then the virus particle with the recombinant
vector can transform plant cell simply by rubbing the
virus onto the surface of a leaf.
 The natural infection process can then spreads the virus
throughout the plant.
206
 The potential of plant viruses as cloning vectors has been
explored for several years.
 One major problem is that the vast majority of plant viruses
have genomes not of DNA but of RNA.
 RNA viruses are not so useful as potential cloning vectors
because manipulations with RNA are more difficult to carry
out.
 Two classes of DNA virus are used to infect higher plants.
 Caulimoviruses and
 Geminiviruses
 The limitation of using viral vectors is that the transgene
cannot be transmitted to subsequent generations.
 The application of viral vectors is important for transforming
vegetatively propagated plants.
207
3.2.2. Application of Transgenic Plants
 Using Biotechnology, scientists can now develop crops with
desirable characteristics quickly and inexpensively by
 Identifying the desired gene in another plant/ animal/
microbes and
 Integrating this desired gene into the recipient plant's
genome, thus creating a transgenic plant.
 Some of the applications of transgenic plants
include
I. Improvement in Agricultural, Horticultural and
Ornamental Value
A. Nutritional Value: Rice grain with vitamin A
 In some countries, rice is the staple food and in poor
communities, it is the only diet.
 As a result, these people suffer from diseases due to
deficiency in vitamins, including vitamin A.
 Vitamin A deficiency:
 makes children vulnerable to infections and causes
208
night blindness
 It is estimated that
 around 400 million people are at risk of vitamin A deficiency,
particularly in Asia and Africa
 deficiency causes 2.5 million deaths annually in children < 5
 500, 000 children go blind each year.
 Supplementation programmes have reduced child
mortality by up to 50% in target areas.
 However this is:
 expensive
 distribution to remote areas is not attainable.
 Through genetic engineering , it was possible to insert
the genes coding for the enzymes that synthesizes a
precursor of vitamin A (β- carotene).
 These genes have been identified and isolated from
another plant Daffodil spp. and a bacterium Erwinia spp.
 The gene was inserted in to a T-DNA and a recombinant
A. tumefaciens was used to transform rice cells.

209
 The trangenic plant cell expresses the precursor on
the grains, which appear yellow

210
B. Delayed-Ripening of Fruits
 The shelf life of fruits and vegetables is also one of
the problems that affects agro-industries.
 There are a number of key metabolic processes that
lead to fruit ripening.
 The gaseous hormone ethylene is one of the
important compounds involved in fruit ripening.
 By blocking the biosynthesis of ethylene, the
process of fruit ripening can be extended.
 Ethylene production can be blocked by inactivating
one or more key enzymes in the biosynthetic pathway.
 This can be achieved by anti-sense mRNA
technology, in which a reverse copy (a
complementary gene) is introduced.
211
212
 The gene coding for the antisense mRNA is inserted in
to the T-DNA region of a Ti plasmid.
 An engineered Agrobacterium is then used to
transform tomato plant cells.
 In the transgenic plant, ethylene production is
inhibited and so the fruit ripening will be very slow.
 When required, fruit ripening can be induced artificially
by supplementing ethylene.
 This delayed ripening helps in exporting tender fruits
such as tomatoes.
 The transgenic tomatoes produce only 2% of the
ethylene that was made by the non engineered
tomatoes.
 These tomatoes are sold as ‘Endless summer
varieties’.
213
 Antisense RNA technology is also similarly applied to
allow maturation of fruits without spoilage.
 As fruits mature, polygalacturonase softens the tissues
of the pericarp and thus make the fruit palatable.
 However, through time the softening spoils the fruit
and make it unattractive to eat.
 To avoid this problem, tomato growers pick the fruit
before ripening.
 But, incomplete maturation results in loss of flavour.
 It is possible to inactivate the genes coding for the
enzyme and allow complete maturation of the fruit on the
transgenic tomato by antisense RNA technology.
 These tomatoes were marketed under the brand ‘Flavr
Savr’ and belong to the first transgenic plants to be
approved safe.
214
C. Tolerance to Abiotic Stress: Drought, Salt,
Osmotic Tolerances
 Plants that are capable of withstanding climate changes
are likely to produce greater yields during severe
weather conditions.
 Gene for tolerance to:

 Drought (Rab genes),

 Salt (e.g. salT gene) and
 osmotic stress (e.g. proBA gene) have been
isolated, cloned and expressed in plants, which
are potential source of resistance to abiotic
stress.
215
 Drought Resistance
 Trehalose (1-  -D-glucopyranosyl-glucopyranoside)
is an important protectant of abiotic stress:
 freezing temperatures
 Osmotic stress (high salinity)
 Heat and dessication
 The gene expressing trehalose is found
in a yeast cell Zygosaccharomyces rouxii.
 Through genetic engineering, it was
possible to develop a drought resistant
transgenic potato by inserting the trehalose
gene.
216
D. Producing Plants Resistant to Pests
and Diseases (Biotic Stress)
 A major challenge for farmers has always been
protection of crops from pathogens like insects,
viruses and fungi.

Insect Resistance
It is estimated that about 15 % of the world’s crop
yield is lost to insects and pests.
Chemical pesticides are used to control the pests.
However, these chemicals have:
 adverse affects on the environment
 Spraying cannot reach vulnerable parts like
217
Biotechnology provides new, and environmentally friendly
ways of dealing with these pests.
Some transgenic tomato, corn, cotton and others
plants have been developed for resistance to pests by
inserting a insect resistance genes.
One of the genes is the cry gene isolated from Bacillus
thuringiensis.
The cry gene expresses an insecticidal crystalline
protein (ICP), a protoxin.
The protein is active against Lepidopteran larvae.
When a plant leaf is ingested by the larvae, the protoxin
is activated by the enzymes in its stomach and is
converted into a lethal toxin that causes paralysis and
death to the larvae.
The use of BT is environmentally friendly, harmless to
other animals, humans and the plants. 218
 Virus Resistance
Virus infection of crops results in:
 Retarded and excessive cell division
 Cell death
With an overall effect of
 growth retardation
 Lowered product yield
 Complete crop failure
 Transgenic plants with virus resistance have been
developed
 One of the characteristics of viral infections is that a
plant cell that has been infected by one virus will not
be reinfected by the same or sometimes other viruses.
 Thus the plant becomes immune to viral infection.
219
 Using genetic engineering techniques, it is possible to
insert a gene coding for a harmless viral coat protein
in to plants.
 When the virus is in contact with such transgenic plant
cells, it will be detect the presence of the coat protein
and it will be tricked in to sensitizing a former infection.
 By employing this system, the first virus resistant
transgenic plant developed was a tobacco plant
carrying the gene coding for tobacco mosaic virus coat
protein.
 Other transgenic plants with coat protein mediated
resistance include:
Rice, potato, wheat, peanut, sugar beet, alfalfa
etc.

220
 Resistance to Fungus
 Fungal infections cause serious damage to crops
every year.
 Genes that code for enzymes that degrade the cell
wall of fungi (chitinase and glucanase) have
been identified from plants and bacteria.
 Transgenic tobacco:
 coding for chitinase from bacteria has
been developed.
 coding for glucanase from barley was
found to be resistant to Rhizoctonia solani.
221
E. Herbicide Tolerant
 Weeds account to 10– 15% of the reduction in crop yield.
 The growth of weeds is controlled by applying herbicides to the
field.
 However, the herbicides do not discriminate between the
weeds from the crop plant.
 Therefore, it is important to develop plants, which are
tolerant to the action of herbicides.
 Glyphosate is a broad spectrum herbicide effective against
76 of the 78 world’s worst weeds.
 Soil inhabiting microbes have the ability to detoxify
glyphosate.
 These microbes possess a gene (gox gene) that codes for
glyphosate oxidase, which converts glyphosate to
glyoxalate and aminomethylphosponic acid.
 A transgenic oil seed rape with the gox gene showed good
222
F. Flower Pigmentation
 Anthocyanins are responsible for the pigmentation of plants.
 The color of the plant is dependant on the nature of
anthocyanin produced
 Pelagonidin 3-glucoside: brick red/ orange
 Cyanide 3- glucoside: Red
 Delphinidin 3- glucoside: Blue to purple
 Desired flower pigment can be produced by manipulating
anthocyanin production pathway.
 Most flowers do not express blue color due to the absence of
an enzyme responsible for synthesis of Delphinidin 3-
glucoside.
 By engineering transgenic plants that carry the gene, blue
flowers can be developed.
223
II. Transgenic Plants as Bioreactors
 Transgenic crops can act as living bioreactors for
inexpensive production of chemicals and pharmaceuticals.
 It has been possible to produce biodegradable plastics,
industrial enzymes, vaccines, carbohydrates, fatty acids,
polypeptides etc

A. Biodegradable Plastics: Polyhydroxybutyrate

(PHB)
 PHB is a biodegradable plastic substrate synthesized from the
precursor acetyl-CoA.
 PHB is mainly produced by bacterial fermentation and the
cost of biodegradable plastics is higher than synthetic
plastics.
224
 Genes encoding the enzymes responsible for PHB
synthesis were transferred to cotton plants by
particle bombardment.
 Adequate PHB plastic was reported in the cotton
fibers.
B. Production of Useful Industrial Enzymes
 Certain industrial enzymes have been produced by
transgenic plants.
 Cellulase and xylanase are important in several
industries:
 Bioethanol (from cellulosic waste)
 Textile and
 Pulp and paper
225
C. Edible Vaccines
 Antigens of several pathogens trigger immunogenic
response when delivered orally.
 The gene encoding the orally active antigenic protein can be
isolated from the pathogen and introduced into the
genome of plants such as banana which are consumed raw.
 The plant part containing the antigen may be fed raw to bring
about immunization.
 Such vaccines produced by transgenic plants are edible
vaccines.
 Transgenic fodder plants that carry a protective antigen
anthrax bacteria can be used as a vaccine against deadly
anthrax.
 Advantages
 They are easy to store and transport without refrigeration.
 The delivery method is also very simple. 226
 3.3. Embryo Transfer and Transgenic Animal
Production
3.3.1. In vitro Fertilization and Embryo Transfer
 When the union between egg cell and sperm cell occurs outside
the body in the culture vessel it is known as in vitro fertilization
(IVF).
 The resulting zygote is cultured in vitro to obtain young embryos,
which ultimately are implanted in the uterus of healthy females to
complete development.
 The implantation of young embryos developed in vitro or obtained
from uterus of donor female into the womb of selected females is
termed as embryo transplantation.
 The babies produced using this approach are known as test tube
babies.
 In medicine, this technique is being applied for couples suffering
from infertility when other methods including assisted reproductive
technologies fail.
227
 Steps in IVF

I. Ovulation
 To maximize the chance for fertilization, large
number of eggs need to be harvested from the
female parent.
 This can be achieved by hormonal treatment for
ovarian hyperstimulation/ superovulation.
 After stimulation, the eggs are allowed to mature
and ovulation takes place.
 The mature eggs are then retrieved by ultrasound
guided needle piercing of the vaginal walls. 228
II. Fertilization, Embryo Development
 Sperm cells collected from the male parent are then
used to fertilize the ovaries by incubating them in a
culture media.
 A normal zygote is selected and cultured for
development to embryo.
 Then, the embryo is cultured on a media until it reaches
morula (32 cell) or blastocyst stage (70- 100
cells).
III. Embryo Transplantation
 Selected embryos (healthy) are then transferred back to
the uterus of the recipient female who can be either
the donor of the egg or a surrogate mother.
 A surrogate is a substitute female body that has been
made receptive by hormone treatment to accept the
embryo and allow its development in the body.
229
230
IV. Embryo Splitting

 A young embryo can be split into 2-4 parts.

 Each of these parts is a zygotic twin, which develop
in to clones of the animal.
 These clones are genetically identical in both their
nuclear and mitochondrial genes.
 Split embryos are placed in an empty zona
pellucida (a thick transparent envelope that
surrounds developing ovum), which is eventually
transferred in to the surrogate mother.
231
 Application of IVF and Embryo Transfer in
Animal Biotechnology
I. It is applicable for rapid rate of multiplication of selected superior
genotypes which can be either :
 naturally existing or manipulated through genetic
engineering.
II. Embryo and sperm cells can be frozen, stored and when
required, it can be used to initiate pregnancy in thousands of
cows.
 Therefore, large number of genotypes of elite animals can be
easily maintained and preserved in limited space.
 It was possible to generate 40, 000 to 50, 000 beef calves
per year with the desired genotype.
III. For production of babies of specified sex.
 If a sperm cell carrying the Y chr. fertilizes an egg, the progeny
is male and if it carries the X chr., the progeny is a female.

232
 Sperm cells carrying Y & X chromosome can be separated
using sephadex column by passing semen, collected from
appropriate male parent, through it.
 Y-carrying sperms are lighter than X- carrying sperms.
 Therefore, Y-carrying sperms are held for longer period in
the column while X- carrying sperms are collected from
the initial fraction of semen collected from the column.
 The appropriate fraction of semen is then used for
artificial insemination of the prospective mother or for
IVF.
 The success in producing babies of specified sex using
this procedure is reported to be close to 90%.
 This technique is employed when a desired animal product
is obtained only from a particular sex (e.g. milk) or when
the yield of a certain product is better in either sex.
233
3.3.2. Transfection Methods in
Animals
 These are techniques applied for the isolation and
transfer of the DNA into cultured animal cells
and embryos to produce various transgenic
animals.
 The gene introduced by transfection is called a
transgene.
 Unlike plants and microbes, animal cell genetic
transformation is complex.
 Alteration of somatic cells would be ineffective
because these cells cannot be passed on to the next
generation. Therefore, genetic manipulations are
234
 Germ lines are linage of cells, which are directly
transferred from the parent to offspring.
 germ lines cells are the most suitable
starting cells.

 Germ lines cells which used for genetic

manipulations which includes:
 Sperm cells;
 Oocytes;
 fertilized eggs;
 early embryo tissue in place
 embryonic stem cell lines in early embryo.
235
 SEVERAL APPROACHES USED TO
INTRODUCE DNA IN TO ANIMAL CELLS.

I. Direct Methods
 Microinjection
 Calcium phosphate precipitation
 Lipofection

II. Retroviral Infection

III.Somatic Cell Nuclear Transfer

236
I. Direct Methods
A.Microinjection:
General Procedures
 Donor female is induced to superovulate using hormone treatment.
 Superovulated eggs are fertilized with sperm cells and embryos
are then collected.
 The transgene is injected in to the male pronucleus using
microinjection assembly.
 Microinjected embryos are cultured in vitro up to morula or
blastocyst stage.
 The surviving embryos are transferred in to the uterus of the
donor or a surrogate female body.
 The embryos develop to full term and give rise to a transgenic
animal.
 Although the method of microinjection is very tedious, microinjection
is the most effective way of gene transfer in animals.

237
B. Calcium phosphate precipitation

238
C. Lipofection

 Sendai virus protein is incorporated into liposome

membrane to facilitate fusion.
239
II. Retroviral Infection

 A virus is engineered to carry the gene of interest.

 As the virus infects the cells it transfers the DNA into the genome
of embryonic cells.
 Through genetic recombination, the gene of interest integrates in
to the genome of the animal cell.

III. Somatic Cell Nuclear Transfer (SCNT)

 In the 1950s Scientists removed the nuclei from frog’s eggs
and replaced it with somatic nuclei from embryo.
 The egg, which carries only the nucleus from the somatic cell
developed in to an adult frog.
 The nuclei from this generation were transferred to other eggs for
the development of more of these frogs.
 These frogs were clones and the process was termed as nuclear
cloning or somatic cell nuclear transfer.
240
 Mammalian SCNT was first successful in sheep, resulting in the formation
of a clone called Dolly.
General Procedure:
 The egg cells from a black head sheep (Egg donor) was
obtained by superovulation and the nuceli were removed
by micro pipette.
 Udder cells of a white head sheep (Nucleus donor) were
isolated and cultured in vitro.
 The udder cells were fused with the enucleated egg cell by
electric shock.
 These fused egg cells (277) were inserted in to different black
headed surrogate mothers.
 Among these egg cells one developed to a healthy lamb,
later on named as Dolly.
 Dolly was white headed and had the same genetic makeup as
the udder cells obtained from the white headed nuclei even
though the egg cell was sourced from a black headed breed.
241
242
 The potential application of SCNT include:

 in animal breeding, for the production of multiple

copies of supreme animals,
 for the preservation of endangered species by
cloning them,
 production of genetically identical laboratory
animals,
 to create homogenous population of cells, tissues
and organs for transplantation and
 to study gene function, genome activation, cancer,
aging and development, etc.
243
 Application of Animal Transfection
A. Improved Productivity
 Transgenic farm animals have been developed with
improved:
 efficiency of meat production (sheep, cattle
and fish)
 wool production (sheep)
 efficiency of feed utilization
 Transgenic Salmon Fish
 A growth hormone transgene was inserted in to
Salmon eggs by microinjection.
 The transgenic Salmon was 10x heavier than the wild
Salmon fish.
244
 Transgenic Sheep
 Cystine is an important amino acid required for
the production of keratin, wool protein.
 However, cystine is inadequately fed to sheep and
bacteria, in the rumen, consume the cystine and
release H2S.
 A transgenic sheep has been developed to carry
a gene from bacteria that synthesizes cystine.
 The products of the gene trap H2S and
synthesize cystine.

245
B. Improved Quality
 Transgenic goats have been developed to produce
milk that have better properties for processing
including
 decreasing clotting time;
 increased curd strength and
 reduced saturated FA.
 Transgenic cattle with an over expressed casein
transgene was able to produce milk with high
content of casein.
 Lactase transgenes that code for lactase, an
enzyme that hydrolyses lactose to glucose and
galactose has been introduced in the mammary
glands.
 The milk produced can be consumed by lactose
intolerant individuals who experience
246
C. Transgenic Animals as
Bioreactors
 Genetic engineering has allowed production of
human proteins and pharmaceuticals from
transgenic animals.
 Production would be commercially viable if
lactating animals are manipulated to collect the
product from the milk.

247
3.4. Bio-fertilizers
 Are organisms that enrich the nutrient quality of soil.
 The main sources of biofertilizers are bacteria, fungi, and
cynobacteria (blue-green algae).
 The most striking relationship that these have with plants
is symbiosis, in which both partners derive benefits.
 Plants have a number of relationship with fungi, bacteria,
and algae, the most common of which are with
mycorrhiza, rhizobium, and cyanophyceae.
 These are known to deliver a number of benefits
including plant nutrition, disease resistance, and
tolerance to adverse soil and climatic conditions.

248
 Biofertilizers will help to solve problems as
increased salinity of the soil and chemical run-offs
from the agricultural fields.
 Bio-fertilizers are eco-friendly organic agro-input
and more cost-effective than chemical fertilizers.
 Bio-fertilizers add nutrients through the natural
processes of nitrogen fixation, solubilizing
phosphorus, and stimulating plant growth through
the synthesis of growth-promoting substances.

End !!!
249
 CHAPTER FOUR

Biotechnology in industry

250
 Recovery and purification of recombinant
proteins
 Recovery of protein: The
method of harvesting a
protein from cloned cells
depends on whether that
protein is found within the
cell or outside the cell. It
is also retrieval of a
protein from broth, cells,
or cell fragments

 Purification – the process

of eliminating impurities
from a sample; in protein
purification, it is the
separation of other
proteins from the desired 251
 The need for purity
 Purity is defined by the general level of protein
contaminants and also by the absence of
contaminants of special interest such as endotoxin,
viruses etc.
 Categories of Proteins.

Category (I)
Proteins produced in bulk as relatively
crudpreparations
 Proteins usually have a wide variety of applications in
food & beverage industries.

Category (II)
 Proteins for therapeutic and /or diagnostic
applications, research purpose.
 Proteins are generally produced in small quantities 252
 Cell Disruption
 Initial recovery of protein depends whether the
protein of interest is intracellular or extracellular.

If intra cellular:
 Achieved by chemical, physical or enzymatic
techniques.

(1) Chemical: Treatment with detergents, antibiotics,

solvents, exposure to alkaline conditions.

(2) Physical: Sonication, Agitation in the presence of

glass beads.

(3) Enzymatic: Treatment with lysozyme, Benzonase

253
254
 Protein Purification Strategies
1. Evaluate an assay for the protein of interest

2. Shortlist a method to have a reasonable source for

that activity

255
 Various techniques
1. Paper Chromatography: Molecules separate as
they move up the paper. The distance that the
molecules travel depends on their size and solubility
in the solvent.

256
2. Thin-Layer Chromatography: Molecules
separate as they move through the silica gel. Thin-
layer chromatography is used to separate small
molecules, such as amino acids.

257
 3. Column Chromatography Operations
There are two ways to run a column:
1. Allow gravity to draw samples and buffers through
the column resin.
2.Use pumps to push a sample and buffers through a
column.
a) Gel-Filtration (Size-Exclusion)
Chromatography:
Used to separate proteins on the basis of their molecular
weight.
The column is packed with a porous resin.
The matrix retards proteins of different sizes for different
periods.
The proteins are collected automatically as they flow out
of the column in tubes held in a fraction collector.
Larger proteins will be eluted first since the smaller 258
proteins travel through the pores of the resin.
Gel Filtration Resin :
When starting protein purification, technicians
sometimes use a gel-filtration (size-exclusion) column
first.
They know the molecular weight of their protein, so
they can often eliminate several contaminant proteins
by a quick run through a sizing column.

259
b) Ion-exchange Chromatography

 In this case, a cation (or alternatively an anion) is attached

to the resin beads.
 Depending upon the electrical properties of the proteins, they
may attach to the column.
 For example, positively charged proteins will stick to a negatively charged
column.
 These proteins can then be removed by washing the column with either a strong
salt solution or changing the pH of the wash buffer.

 Anion exchangers such as DEAE ( Diethyl amino ethyl) are

used.
Attraction of proteins at a pH above the isolectric point of
the protein.
 Cation exchangers such as CM ( Carboxy methyl) are
used.
Attraction of protein at a pH below the isoelectric point of
the protein. 260
Ion – exchange Chromatography,….Cont’d

261
Ion-Exchange Chromatography
Ion Exchange
Resin
Resins are
manufactured with
ions attached. The
ions present a
certain degree of
positive or negative
charge, depending
on the buffer pH.

262
 c) Hydrophobic chromatography

Principle

 Separation based on the degree of protein hydrophobicity

 Hydrophobic residues in proteins: Valine, Leucine ,

Isoleucine, Tryptophan etc.

 Resins: Cross linked agarose gels to which hydrophobic

groups have been covalently attached. May contain a
phenyl or octyl hydrophobic groups.

263
264
 d) Affinity chromatography
 In this type of chromatography, a compound with a special affinity
for the protein of interest is attached to the resin. For example, in
immunoaffinity chromatography antibodies to a specific protein
(or its domain) are used as the specialized compound.

 The resin is then packed into a column. When a mixture of

proteins is passed through the column, only those proteins with
special affinity for the compound will stick to the column. All the
other proteins will pass through the column. Once the non-specific
proteins are eluted, proteins of interest that have stuck to the
column can be eluted (fraction #3). These proteins can be
removed by changing the ionic strength of the solution (so
affecting the strength of binding of the protein to the column).
Alternatively the special compound can be added to the elution
solution and the equilibrium will change so that the protein will no
longer stick to the column.

265
Affinity chromatography,…….Cont’d

266
 Other form of column chromatography

1. Open Column Chromatography

Also called gravity-flow

chromatography

2. Fast-Performance Liquid
Chromatography (FPLC)
FPLC: Pumps push the buffer or sample
through tubing, into and through the column.
As fractions come off the column, they are
run through a spectrophotometer that
determines the protein concentration of the
sample.

267
3. High-Performance Liquid Chromatography (HPLC)
Greatly improved ability to separate, purify, identify, and
qualify samples.

Resins Used in Column Chromatography

There are several types of resins available. For ion-exchange
chromatography, resins have either positive or negative
charges at a given pH.

268
 Chromatographic Modes of Protein Purification
Chromatographic Mode Acronym Separation Principle
Non-interactive modes of liquid chromatography

Size-exclusion chromatography SEC Differences in molecular size

Slalom chromatography (for DNA) - Diff. in length and flexibility

Interactive modes of liquid chromatography

Ion-exchange chromatography IEC Electrostatic interactions
Normal-phase chromatography NPC Polar interactions
Reversed-phase chromtography RPC Dispersive interactions
Hydrophobic interaction HIC Dispersive interactions
chromatography
Affinity chromatography AC Biospecific interaction

Metal interaction chromatography MIC Complex w/ an immobilized

metal
269
Enzyme Technology/Enzymology

Industrial application of Enzymes

270
 Enzymes are useful

 They are catalysts so they make reactions easier. This

increases productivity and yield
 As catalysts they are not consumed by the reaction.
They may be used over and over again.

 Enzymes show specificity to the reaction they control.

 Enzymes are sensitive to their environment so they can

be controlled by adjusting the temperature, the pH or
the substrate concentration
271
 Enzymes – the key to it all

 Almost all chemical reactions that take place inside living things
are controlled by enzymes.

Enzymes are large molecules found in living things that

act as biological catalysts.

 Catalysts are chemicals that speed up reactions without getting

used up.

 Research has shown that the Enzymes are not

living - they are just
shape of enzymes is the key
molecules.
to their catalysis.

272
 A catalyst will Speed up a reaction without being used up

 An enzyme is … A biological catalyst

 Enzymes are obtained from …
Living things, usually microbes such as bacteria or yeast

 Enzymes only work if these conditions are correct …

Temperature (usually 20-40oC) and pH.

 The important part of an enzyme is called … The active site

 The words used to describe the importance of shape are ..
Lock and key

 An enzyme that has lost its shape is denatured

273
 Sources of Enzymes

 Biologically active enzymes may be extracted from

any living organism:

 Of the hundred enzymes being used industrially,

 over a half are from fungi

 over a third are from bacteria with the

remainder divided between animal (8%) and
plant (4%) sources.

274
Microbes are preferred to plants and animals as
sources of enzymes because:

 They are generally cheaper to produce.

 Their enzyme contents are more predictable and

controllable.

 Plant and animal tissues contain more potentially

harmful materials than microbes, including phenolic
compounds (from plants).
275
 Fungal Enzymes

Enzyme Sources Application

a-Amylase Aspergillus E Baking

Catalase Aspergillus I Food
Cellulase Trichoderma E Waste
Dextranase Penicillium E Food
Glucose oxidase Aspergillus I Food
Lactase Aspergillus E Dairy
Lipase Rhizopus E Food
Rennet Mucor miehei E Cheese
Pectinase Aspergillus E Drinks
Protease Aspergillus E Baking

E: extracellular enzyme; I: intracellular enzyme

276
 Bacterial Enzymes

Enzyme Sources Application

a-Amylase Bacillus E Starch

b-Amylase Bacillus E Starch
Escherichia
Asparaginase I Health
coli
Glucose
Bacillus I Fructose syrup
isomerase
Penicillin
Bacillus I Pharmaceutical
amidase
Protease Bacillus E Detergent

277
 IMMOBILISED ENZYMES

 Enzymes are expensive, they should be utilized in an efficient

manner
 As catalytic molecules, enzymes are not directly used up.
After the reaction the enzymes cannot be economically
recovered for re-use and are generally wasted
 This enzyme residue remains to contaminate the product and
its removal may involve extra purification costs.
 Simple and economic methods must be used to separate the
enzyme from the reaction product

278
Separation of enzyme and product using a two-phase system;

* One phase containing the enzyme

* The other phase containing the product

This is known as
IMMOBILISATION

 The enzyme is imprisoned within its phase allowing its re-use or

continuous use the separation prevents the enzyme from
contaminating the product

279
 Methods of Immobilisation

 Adsorption
 Covalent binding
 Entrapment
 Membrane
confinement

280
Entrapment Immobilization is based on the localization of an enzyme
within the lattice of a polymer matrix or membrane.
- retain enzyme
- allow the penetration of substrate.
Entrapment
- Matrix Entrapment - Membrane Entrapment
(microencapsulation)

281
Surface immobilization

According to the binding mode of the

enzyme, this method can be further sub-
classified into:

 Physical Adsorption: Van der Waals

Carriers: silica, carbon nanotube,
cellulose, etc.
Simple and cheap, enzyme activity
unaffected.

 Ionic Binding: ionic bonds

Similar to physical adsorption.
Carriers: polysaccharides and synthetic
polymers
having ion-exchange centers.
282
 Cross-linking is to cross link enzyme molecules
with each other using agents such as
glutaraldehyde.

 Features: similar to covalent binding.

 Several methods are combined.

283
 Matrices for Enzyme Immobilization

 Inert.
 Physically strong and stable.
 Should be cheap enough to discard.
 Better if it could be regenerated after the useful lifetime of
the immobilized enzyme.
 The surface available to the enzyme.
 More desirable properties:
* Ferromagnetism (e.g. magnetic iron oxide, enabling
transfer of the biocatalyst by means of magnetic fields).
* Catalytic surface (e.g. manganese dioxide, which
catalytically removes the inactivating hydrogen peroxide
produced by most oxidases).
* Reductive surface environment (e.g. titania, for
enzymes inactivated by oxidation).

284
Sources of Antibiotics and Secondary Metabolites

Antibiotics
Compounds that kill or inhibit the growth of other microbes
Typically secondary metabolites
Most antibiotics in clinical use are produced by filamentous fungi
or actinomycetes
Still discovered by a laboratory screening process
Assayed for products that inhibit growth of test bacteria

285
Primary Metabolite
– Produced during exponential growth
– e.g., alcohol

Secondary Metabolite
 Produced during stationary phase
 Not essential for growth
 Formation depends on growth conditions
 Produced as a group of related compounds
 Often significantly overproduced
 Often produced by spore-forming microbes during sporulation

286
Secondary Metabolite Production

287
 Fungal secondary metabolites
Non-ribosomal peptides (NRPS) Terpenes

b-lactam antibiotics Trichodiene - toxin

Cyclosporin – immunosuppressant Aristolochene - toxin
Echinocandin – antifungal drug

Polyketides (PKS)

Lovastatin – cholesterol lowering agent

Aflatoxin – carcinogen

Indole alkaloids

Ergotamine – migraine treatment

– control of post partum bleeding

288
 Plant Secondary Metabolites

 Plants make a variety of less widely distributed

compounds such as morphine, caffeine, nicotine,
menthol, and rubber.

 Secondary metabolites or secondary compounds are

compounds that are not required for normal growth and
development, and are not made through metabolic
pathways common to all plants.

 Most plants have not been examined for secondary

compounds and new compounds are discovered almost
daily.
289
 Secondary compounds are grouped into classes based on similar
structures, biosynthetic pathways, or the kinds of plants that make
them.

 The largest such classes are the alkaloids, terpenoids, and

phenolics.

 Secondary compounds often occur in combination with one or

more sugars.

 These combination molecules are known as glycosides. Usually

the sugar is a glucose, galactose or rhamnose.

 But some plants have unique sugars.

 Apiose sugar is unique to parsley and its close relatives.

290
 Functions of Secondary Compounds
 The most common roles for secondary compounds in plants are
ecological roles that govern interactions between plants and other
organisms.

 Many secondary compounds are brightly colored pigments like

anthocyanin that color flowers red and blue. These attract
pollinators and fruit and seed dispersers.

 Nicotine and other toxic compounds may protect the plant from
herbivores and microbes.

 Other secondary compounds like rubber and tetrahydrocannabinil

(THC) from cannabis plants have no known function in plants.

291
 Some secondary metabolites are produced for easily
appreciated reasons, e.g.
 As toxic materials providing defense against predators.
 As volatile attractants towards the same or other species.
 As coloring agents to attract or warn other species.

 The most important building blocks employed in the

biosynthesis of secondary metabolites are derived from:
 Acetyl coenzyme A (acetyl-CoA)
 Shikimic acid
 Mevalonic acid
 1-deoxyxylulose 5-phosphate
 Amino acids

292
 Alkaloids
 Alkaloids generally include alkaline substances that have
nitrogen as part of a ring structure.

 More than 6500 alkaloids are known and are the largest class
of secondary compounds.

 They are very common in certain plant families, especially:

 Fabaceae – peas and beans
Asteraceae – sunflowers
Papaveraceae – poppies
Solanaceae – nightshade, tomato
Apocynaceae – dogbanes
Asclepiadaceae – milkweeds
Rutaceae - citrus

293
Terpenoids
 Terpenoids are dimers and polymers of 5 carbon precursors called
isoprene units (C5 H8).
 Terpenoids often evaporate from plants and contribute to the haze
we see on hot sunny days.
 They are expensive to make; they often take 2% of the carbon fixed
in photosynthesis; carbon that could otherwise be used for sugars.
Phenolics
 Compounds that contain a fully unsaturated six carbon ring linked
to an oxygen are called phenolics.
 Salicylic acid (basic part of aspirin) is a simple phenol.
 Flavonoids are complex phenolics. They are often sold in health
food stores as supplements to vitamin C.
 Anthocyanins are a type of flavonoid that give flowers red and blue
pigments.
294
 The need for new antibiotics
New antibiotics
Originally, screening for new antibiotics was based on testing the
bacterial or fungal extracts.

A number of drugs developed in the middle of the 20th century still

remain among the best.

These include aminoglycosides, cephalosporins, tetracyclines,

macrolides, etc.

There is now a renewed interest in screening natural sources

(especially, non-traditional, e.g. marine microorganisms)

295
 New antibiotics: genome-based approaches
3000

2500

2000

1500

1000

500

0
Genome Essential Genes Targets for
Current
Antibiotics

Genomics holds a good potential for identifying new drug targets.

296
 !!!
N D
E
297
 CHAPTER FIVE

Environmental biotechnology

298
 Environmental biotechnology

What was environmental biotechnology?

 Simple and traditional definition: use, in a controlled manner,
of microorganisms to degrade wastes.

 Solving environmental problems through biotechnology; e.g.

biosensor, Bio Microelectronics and Nanotechnologies,
Biotreatments, etc.

 International Society for Environmental Biotechnology,

since 1992. Two streams:
(1) microbial biotechnology for environmental improvement
(sewage treatments and bioremediation) and
(2) chemical engineering related to the environment. From waste
treatment to bioremediation.
299
 What was environmental biotechnology?,…..Cont’d

 Use of molecular techniques to protect the environment,

including Risk assessments of GMOs

 Renewable energy and resources: engineering plants for

the production of clean energy, biofuel, biomass, and animals
for food production, etc.

 Environmental Biotechnology is the multidisciplinary

integration of sciences and engineering in order to utilize the
huge biochemical potential of microorganisms, plants and
parts thereof for the restoration and preservation of the
environment and for the sustainable use of resources

300
 5.1.1. Waste and wastewater treatment
A) Bioremediation (site restoration) and Biotechnology for Waste
Treatments:
 Ocean ranching for stock restoration (e.g. cultured salmon,
grouper and abalone released to the sea or artificial reef).
 Recovering of damaged sites to a “clean” or less harmful site
after dredging.
 Remove chemicals using biological treatments on site (in situ) or
ex situ.
 Chemicals: heavy metals, trace organics or mixtures.
 Bacterial or fungal degradation of chemicals: Engineered
microbes for better and more efficient removal of chemicals on-
site
301
 1) Redox Clean-Up Reactions

 Anaerobic or aerobic metabolism involve oxidation and

reduction reactions or Redox reactions for
detoxification.
 Oxygen could be reduced to water and oxidize organic
compounds.
 Anaerobic reaction can use nitrate.

 In return, biomass is gained for bacterial or fungal growth.

302
2) Problems with bioremediation

 Work in vitro, may not work in large scale. Work well in the
laboratory with simulation, may not work in the field.
 Alternatively, select adapted species on site (indigenous
species) to remediate similar damage.
 Most sites are historically contaminated, as a results of the
production, transport, storage or dumping of waste.
 They have different characteristics and requirements.

303
3) Use of bacteria in bioremediation:-

 Greatly affected by unstable climatic and environmental factors

from moisture to temperature.
 For examples, petroleum hydrocarbon degrading bacteria do
not work well less than one degree centigrade.
 These microbes are usually thermophilic anaerobic.

 Fertilizers are needed.

 Seeding or bioaugmentation could be useful too.
 They contain monooxygenases and dehydrogenases to break
down organic matters including most toxic substances.
304
 4) Use of fungi in bioremediation:-

 Lipomyces can degrade paraquat (a herbicide).

 Rhodotorula can convert benzaldehyde to benzyl alcohol.

 Candida can degrade formaldehyde.

 Gibeberella can degrade cyanide.

 Composting can be used to degrade household wastes

305
5) Phyto-remediation
 Effective and low cost

 Soil clean up of heavy metals and organic compounds.

 Pollutants are absorbed in roots, thus plants removed could be

disposed or burned.

 Sunflower plants were used to remove cesium and strontium

from ponds at the Chernobyl nuclear power plant.

 Transgenic plants with exogenous metallothionein (a metal

binding protein) used to remove metals.

306
 B) Waste water treatments

 Bioremediation of water or groundwater recovered from

polluted sites.

 Ex situ: As many bacteria work better in controlled conditions,

e.g. anaerobic, higher temperature, effluent (sewage treatment) or
solid materials (composting) can be treated with bacteria to
decompose organic matters.

 Primary treatment: screening and emulsification.

 Secondary treatments: Nutrient removal and chemical removal.

307
1) Nutrient removal

 Phosphate removal by polyphosphate accumulating

organisms and glycogen accumulating organisms.
 Nitrogen removal by Nitrosomonas which denitrify
nitrite to nitrogen gas. Anaerobic ammonium oxidation
is also important.
 Algae could absorb many nutrients and pollutants.
Dunaliella, Chlorella and Spirulina are valuable species.

308
 2) Dye removal and chemical removal
Azo-dye (N=N) removal

Sensitive to redox and anaerobic treatments can decolorize

azo dyes

Specific reductase enzymes are needed to detoxify the dye

after discoloration

Ammonia removal
 Chemical treatment or biological treatment
e.g. Candidatus, Brocadia, Anammoxidans

309
5.1.2. Biosensors /monitor pollution/

 Biosensors are detecting devices that rely on the specificity of

cells and molecules to identify and measure substances at
extremely low concentrations.
 Measurement of mutagenic activity (microtox and mutatox tests
with lux gene from Vibrio)
 Detection of pathogens by multiplex-PCR
 Detection of toxins (Ciguatoxin)
 A biosensor is composed of a biological component, such as a cell,
enzyme or antibody, linked to a tiny transducer.
 Biosensors can, for example,
 measure the nutritional value, freshness and safety of food.
 locate and measure environmental pollutants.
 detect and quantify explosives, toxins and biowarfare agents.
310
1) Bio Detection Systems
 CALUXR Bioassay
 A sensitive bioassay for exposure to
dioxins and related compounds

 Synthetic gene promoter was created

and linked to a reporter gene which
gives color when the gene promoter is
turned on.

 The synthetic gene promoter contains

multiple cis-acting elements
responsible for dioxin (DRE) and
dioxin receptor (Ah receptor) binding.

 The reporter gene is transfected into a

cell-line for the bioassay.

311
 2) Pathogen detection

 Bacteria: coli form bacteria, salmonella, Legionella, Vibrio,

etc.
 Virus: Influenza, hepatitus, polio, etc.
 Algae: dinoflagellates, diatoms, toxic algae, ciguatoxin, etc.
 Multiplex technology is being developed: one run for many
pathogens.
 Collection with minimal amount of samples: water, soil, or air.
 Use PCR or real-time PCR techniques

312
 3) Stress Proteins
 Metallothionein for exposure to heavy metals
 Cytochrome P450 (CYP) IA1 for exposures to trace organics
 Vitellogenin (an egg yolk protein) for exposure to
environmental estrogens
 Heat shock protein for general stress conditions
 These biomarkers are NOT biomarkers of toxic effects.
 They are biomarkers of exposures. = Still controversial
 Biomarkers have biological relevance and usually less
expensive than chemical analyses. Data could be diagnostic
and indicative.

313
5.2. Biotechnology in the energy sector
5.2.1. Bio-fuel and biodiesel production

 Plant-derived fuels: plant species for:

 Hydrocarbon (oil) production, e.g. rape-seed, sunflower, olive,
peanut oils. Or
 Ethanol production of sugars (or cellulose) derived from plants.

 Conversion of used cooking oil to bio-fuel (called bio-diesel).

 Biogas: gases from composts or landfill, but methane is a green

house gas

314
a) Bioethanol and biofuel cell:
 Sugar cane, sugar beet wastes, high starch material (cassava, potatoes, millet) to
be hydrolyzed by starch hydrolyzing enzyme to convert sucrose or glucose to
ethanol. Mainly used in Brazil.
 Corn ethanol: 22% less carbon emission, used in the US.
 Bio-diesel: 68% less carbon emission; oils from soybean (US) or canola oil
(Germany)
 Cellulosic ethanol: 91% less carbon emission, but difficult to change cellulose to
ethanol
 Hydrogen energy however is the trend of future renewable energy without
carbon emission: a journey to forever…….
 Problem is how to generate the hydrogen; too costly with conventional chemical
methods or reverse osmosis.
315
 b) A Journey forever?
 Various bacteria and algae, for
example Escherichia coli,
Enterobacter aerogenes, Clostridium
butyricum, Clostridium
acetobutylicum, and Clostridium
perfringens have been found to be
active in hydrogen production under
anaerobic conditions.

 The most effective H2 production is

observed upon fermentation of
glucose in the presence of
Clostridium butyricum

316
General characteristics of chemical and biological fuel cell
 
  Chemical Fuel Cell  Biological Fuel Cell
Catalyst Noble Metals  Microorganism /
enzyme
pH Acidic Solution (pH<1) Neutral Solution pH
7.0-9.0
Temperature  over 200 ° C Room Temperature
22-25 ° C
Electrolyte Phosphoric-acid Phosphate Solution 
Capacity High Low
Efficiency 40 - 60 % over 40 %
Fuel Type Natural gas, H2, etc. Any Carbohydrates
and hydrocarbons

317
 CHAPTER SIX

BIO-SAFETY AND SECURITY ISSUES

318
 General concerns about biotechnology applications
Medicine :
• Delivering vaccines

• Gene therapy
Agriculture:
• Resistance to insects or viruses – reduced need for insecticides

• Tolerance to herbicides

• Reduced need for irrigation

• Resistance to frost (tomatoes), salinity

Bioremediation
 bacteria that consume e.g spilled oil
319
 a) What are Biosafety and Biosecurity?

 Biosafety refers to the “development and implementation of

administrative work practices, facility design and safety
equipment to prevent the transmission of biologic agents to
workers, other persons or the environment.”
Biosecurity measures to protect the release of high consequence
microbial agents, biological pathogens, toxins, critical information,
pests or diseases as a result of theft or misuse.

320
b) Risk assessment and management of biotechnology
products

 Identify the Risks (Risk Assessment)

 Minimize the risks
 Safe Operating Procedures
 Monitor the Risks
 Spill / Exposure contingency plans
 Inductions
 Training
 Good laboratory practice
321
c) Bio-safety protocols

 Microorganisms could be removed from a laboratory and

cultured with no-one knowing.

 Bioterrorism - real threat to the modern world.

 Biosecurity protocols should be included in the development

of any new procedures.

 Awareness of practices in the laboratory are crutial.

322
 Social and legal issues
a) Public awareness about biotechnology products
 While modern biotechnology may be considered as one of the
main economic development forces for the twenty-first
century,

 It equally presents far-reaching legal, moral and ethical

implications for society.

 Central to the application of biotechnological techniques to a

wide range of industries is gene technology - a controversial
and emotive subject.

323
Genetic Engineering,....

 Has the potential to improve human health, nutrition and

comfort

 but…….

 It carries social, ethical and environmental risks, many of

which may be presently unforeseen

• How can we manage such a huge and complex issue?

324
Genetic engineering – Are there negatives??

Unknown
No long
side
term
effects
testing Gene Toxins
pollution Antibiotic
resistant
Crop failure Allergies
bacteria

325
b) Ethical Issues
A wide variety of social and ethical issues are
associated with:
 biotechnology research,
 product development and
 commercialization.
 Some of the main Ethical issues concerning rDNA
technology will be examined in some detail below:

326
 1) Genetic modification and food uses
 Genetic engineering to food production is intended to enhance
the useful and desirable characteristics of the organisms and to
eliminate the undesirable.
 The overall aim of the food industry, with respect to genetic
engineering, will be:
 To improve the quantity and
 To increase the quality and properties of existing food
productions,
 To produce new products and, of course, to improve financial
returns.
 The consumer has always shown a willingness to pay more for
better and more convenient products and to reject products that
do not meet their expectations.

327
 While the public accepted medical products produced from GMOs,
 they are much less willing to accept such procedures with food.
 Genetic engineering is seen as ‘unnatural’ and unnecessary in food
production. While scientific opinion is well respected in medical
matters by the public, it is often perceived, in matters of food, as
purely commercially driven.
 The safety of the human food supply is based on
 the concept that there should be a reasonable certainty that no
harm will result from its consumption.
 Public confidence must be achieved for the success of any new
technology
328
Micro-organisms are used to:
• Turn milk into cheese and yogurt.
• To ferment beer and wine.
• Yeast is used in bread to make it
rise.

Yes Biosecurity risks are

associated with these
products as well!!!

329
2) The applications of human genetic research
 genetic disorders of humans result from a mutation in single genes,
 Results from the Human Genome Project, give, in some cases,
hope for alleviation and perhaps cure of the defect, but….
Areas of public concern in human genome research
 Confidentiality of testing and screening results
 Scope of genetic testing and screening
 Discrimination
 Commercial exploitation of human genome data
 Eugenic pressures
 Effects of germ-line gene therapies on later generations

330
3) Gene therapy:
 Gene therapy is subject to greater oversight than virtually all
other therapeutic technologies.
 Thousands of patients have now received somatic cell (non
reproductive cell) gene therapies targeted at life-threatening
genetic diseases, cancer and AIDS.
4) Germ-line gene therapy
 the academic and industrial research communities have
observed gene therapy procedures that would affect the germ-line
cells—the egg and sperm—that pass on genetic composition.

331
 5) Stem Cell Research

Non specialized cells

that have the ability to
The Debate produce specialized cells
Continues!!! for various tissues in the
body.

Embryonic stem cells

are originally harvested
from an embryo.

Expected advances in
the treatment of cancer,
Biosafety protocols must be paramount Parkinsons and
Alzheimers
332
6) Cloning
 Cloning is a generic term for the replication in a laboratory of
genes, cells or organisms from a single original entity.
 Human reproductive cloning would involve taking the nucleus of
a somatic cell (a body cell that is neither an egg nor a sperm) of a
person and inserting it into an unfertilized egg from which the
nucleus has been removed.
 The egg containing the somatic cell nucleus is then implanted into
a woman’s uterus.
 Reproductive cloning is too dangerous and raises far too many
ethical and social questions to be undertaken.
333
Looking to the future,….
 Biotechnology will play a major role in the continued search for
solutions to the many problems that will affect the society of
tomorrow, namely;
 health,
 food supply and
 a safe biological environment.

 Continued scientific research will be paramount to

achieving these ends.

334
E ND

335