Genetic recombination is the process by which bacteria exchange genetic material. It occurs through transformation, transduction, and conjugation. Transformation involves the uptake of naked DNA by bacteria. Transduction involves the transfer of DNA between bacteria by bacteriophages. Conjugation involves the transfer of DNA through direct contact between "male" and "female" bacteria through plasmids. These processes increase genetic diversity and allow for the spread of traits like antibiotic resistance.

1. GENETIC RECOMBINATION
-BY N. B. BANARASE,
Associate Professor
APOLLO COLLEGE OF PHARMACY,
ANJORA, DURG (C.G.)

2. What is Genetic Recombination? How does it
takes place in Bacteria?
 Genetic Recombination is the mixing of
the genetic materials of a bacterial species into
new combinations.
 Genetic Recombination in bacteria takes place
by-
1) Transformation
2) Transduction &
3) Conjugation

3.  It is the process of transfer of
genetic material through free DNA.
 It was first discovered by Griffith in
1928.
 In this experiment he injected the
mixture of Live non-capsulated (R)
Pneumnococci & Heat killed
capsulated (S) Pneumnococci to
Mice.
 Actually both are non-virulant to
mice—so Mice should be alived.
 But it killed?

4.  When bacteria isolated from mice---They found to be
Live capsulated Pneumnococci!
 But they were already killed. Then how could they
gets life again.
 Further studies found that, some genetic material
were transfers from to .
Which contain information of .
 In 1944, Avery, MaCleod and McCarty found that, the
material transferred was DNA.

5. Conjugation
 It is the process of transfer of genetic
material where ‘Male’(Donor)
bacterium makes physical contact
with ‘Female’ (Recipient) bacterium.
Which contains ‘Plasmid’
(having infectious character)
Plasmid is absent.
 So, after its transfer, female bacterium
converts into male bacterium!!!!
 It was first described by Lederberg &
Tatum (1946) using E.Coli strain (K12)

6.  In this process, the plasmid in
male bacterium replicates &
transfers to the female
bacterium through fimbria “Sex
Pillus”.
 Plasmid transferred to female
bacterium contains infectious
characters and sometimes some
portion of host DNA (i.e. Male
DNA).
 The DNA of host is then
combines with the DNA of
recipient effecting genetic
recombination.

7. The F Factor
 The plasmid responsible for conjugation is called “F
(Fertility ) Factor”.
 It contains the genetic information for synthesis of sex
pilus but is devoid of information like drug resistance.
 Cells containing plasmid (Male) called as F+ cells and
females as F- cells.
Hfr Cells
 In some cells fertility factor is present in its ‘Integrated
State” called as “Hfr” Cells.
 It means F-factor of donor integrated with self DNA. So
resulting factor contain the whole genome of this
bacteria. But such cell when conjugate with F- cells ---it
produce very less chances to convert F- to F+

8. This conversion of F+ cell into Hfr state is reversible when F factor reverts from the
integrated state to the free state.

9.  Transfer of portion of DNA from one bacterium to another
by is known as “Transduction”.
 What is bacteriophage?
 -These are the viruses that infect the bacteria.
 Types of transduction:-
 A) Generalized transduction &
 B) Specialized transduction

10. GENERALIZED TRANSDUCTION
 Generalized transduction is the process by which any bacterial gene may
be transferred to another bacterium via a bacteriophage, and typically
carries only bacterial DNA and no viral DNA.
 If bacteriophages undertake the lytic cycle of infection upon entering a
bacterium, the virus will take control of the cell’s machinery for use in
replicating its own viral DNA. If by chance bacterial chromosomal DNA
is inserted into the viral capsid which is usually used to encapsulate the
viral DNA, the mistake will lead to generalized transduction.
 Further the virus having bacterial DNA gets replicates and multiplied
within bacterial cell.
 After lysis of bacterial cell the bacteriophages will infect to another
bacteria and released its genetic material which results into
recombination of new bacterial DNA with resident bacterial DNA.

11.  In Specialized transduction, the phage DNA
integrated with bacterial DNA (Prophage).
 This prophage gets replicate within the bacterium
into multiple copies (i.e. new virus particle)
containing viral genome along with bacterial.
 After lysis of bacterial cell, new virus particle gets
infect to another bacterium and transfer their
genetic material which results into gene
recombination.
SPECIALIZED TRANSDUCTION

12. PROTOPLAST FUSION
 What is protoplast?
 - A protoplast is a plant,
bacterial or fungal cell that had
its cell wall completely or partially
removed using either mechanical
or enzymatic means.
 Protoplast can be obtained from
any plant species.
 Protoplast fusion, is a type
of genetic modification in plants
by which protoplasts of two
distinct species of plants are fused
together to form a new hybrid
plant with the characteristics of
both. It is also called as “somatic
fusion”.

13. METHODS OF PROTOPLAST FUSION
 SPONTANEOUS FUSION
 -During the enzymatic degradation
of cell walls some of the adjacent
protoplasts may fuse together to
form homocaryons (protoplasts
having genetically identical nuclei of
one species). These plurinucleate
cells sometimes contain 2-3 nuclei, a
phenomenon attributed to
expansion and subsequent
coalescence of the plasmodesmatal
connections between the cells.

14.  INDUCED FUSION
 --Fusion of freely isolated protoplasts from different
sources with the help of inducing chemical agents or
other means are known as “induced fusion”. Normally,
isolated protoplasts do not fuse with each other
because the surface of the isolated protoplasts carries
negative charge around the outside of plasma
membrane and thus is a strong tendency for
protoplasts to repel one another due to their same
charges.
 --The isolated protoplast can be induced fuse by
 A) Mechanical means- where protoplasts are fused by
bringing them closer to each other by perfusion
micropipette.
 B) Chemofusion- Fusion is brought by using chemical
agents like sodium nitrite, PEG, polyvinyl alcohol etc.
 C) Electrofusion- Here electric field is used for fusion.

15.  After fusion gets took place, it is
checked with respect to its
pigmentation, presence of
chloroplast, nuclear staining etc
under the microscope which
always shows differentiation
from parent cells.
 The selected fused protoplast
cell having hybrid characters are
allowed to grow on nutrient
media which develops into
callus and subsequently to
plant.

16. Advantages of Protoplast Fusion
 This technique leads to the production of new genetic
variations.
 It provides a potential tool for combining the genome of
different genera and species.
 Through protoplast fusion, some useful genes for various
characters can be transferred like disease resistance,
nitrogen fixation etc.

17. GENE MUTATION
 What is gene mutation?
 - Genes are segments of DNA located
on chromosomes . A gene mutation is defined as an
in the structure of gene.
 The substances that brings such mutations are termed
as .
 Such mutations can takes place during replication,
transcription (formation of m-RNA in nucleus)and
translation (Protein synthesis from m-RNA).
 Altering genes most often results in
which can worsen to various diseases also.

18. TYPES OF GENE MUTATION
 1) Point Mutation-
 The replacement of one base pair by another results in
“Point Mutation”. They are of two types-
 a) Transitions: In transitions, purines or pyrimidine is
replaced by another.
 b) Transversion: In transversion, purine is replaced by
pyrimidine or vice versa.
 2) Frame shift Mutation:
 In this mutation either one or more base pairs are
inserted in or deleted from the DNA respectively
causing insertion or deletion mutation

19. CONSEQUENCES OF POINT MUTATIONS
 Change in single base sequence in point mutation may cause one of followings-
 a) Silent mutation: The codon (of mRNA) containing the changed base may code for
the same amino acids. For instance UCA codes for serine and change in the third
base i.e. UCU still codes for serine. So, no detectable effects occurs in silent
mutation.
 b) Missense mutation: In this case, the change in base may code for a different
amino acid. (e.g. UCA-Serine and ACA-Threonine). So, change in amino acid may be
acceptable, partially acceptable or unacceptable regarded to function of protein
molecule.
 c) Nonsense mutation: sometimes, the codon with the altered base may become a
termination (nonsense) codon. (e.g. change in second base of serine codon UCA
may result in UAA). This altered codon acts as stop signal and causes termination of
protein synthesis.
 CONSEQUENCES OF FRAMESHIFT MUTATIONS
 - The insertion or deletion of base in gene results that the protein synthesized will
have several altered amino acids and/or prematurely terminated protein.

20. HYBRIDOMA TECHNOLOGY
 Hybridoma Technology is a technology forming hybrid cell lines
by fusing an antibody-producing B-cells with myeloma (cancer
cell having characteristic of continuous growth) cells that is
selected for its ability to grow in tissue culture.
 Why there is need of technology called as “Hybridoma”?
 When we use conventional methods for the preparation of
antisera containing antibodies against antigens. It contains
polyclonal antibodies which can not produce specific reactions.
 So, there is a need of antisera which contains monoclonal
antibodies.
 And such antibodies preparations are possible through
“Hybridoma Technology”.
 It was discovered by George Kohler and Cesar Milstein in 1975.

21. •The main function of B-cells are to produce antibodies that
binds to specific antigen.
•Each B-cell have unique receptor on its surface that binds to
only specific antigen.
•Plasma B-Cells when expose to antigen it secretes large
amount of antibodies (Other are Memory B-cell)

22.  1) Laboratory animals like mice are first exposed to the
antigen that an antibody is to be generated against it
generally by giving series of injections of an antigens.
 2) After specific period when antibodies formation
takes place, B-cells of spleen are isolated.
 3) B-cells are then fused with myeloma cells (myeloma
cells are selected beforehand to ensure that they are
lack of hypoxanthine-guanine
phosphoribosyltransferase i.e. HGPRT gene, making
them sensitive to HAT medium)by using electrofusion
technique.

23.  4) Fused cells are incubated in HAT (Hypoxanthine-
aminopterine-thymidine) medium for 10-14 days.
 ** aminopterine blocks the pathway that allows for
nucleotide synthesis. Hence, unfused myeloma cell dies
as they cannot produce nucleotide due to lack of HGPRT.
 5) Also unfused B-cells dies due to short life span.
 6) so only B-cells—myeloma hybrids survive (due to
presence of HGPRT from B-cells) which can synthesize
antibodies.
 7) These hybrids are then allowed to grow in multi-well
plates to such an extent that each well contains only one
hybrid.
 8) After incubation to specific period, antibodies
produced in each well is screened for specificity by
putting them into the well plate having an specific
antigen bound to its surface.

24.  9) Specific antibodies producing hybrids binds to selected
antigen are selected while others are discarded.
 10) Desired antibody producing hybrids are then cultured
on specific culture media in culture flask to produce
monoclonal antibodies.
Applications:
The antibodies produced by hybridoma technology have
been widely used for a variety of purposes. These includes
early detection of pregnancy, detection and treatment of
cancer, diagnosis of leprosy and treatment of
autoimmune diseases.

25. Polyclonal antibodies Monoclonal antibodies
Inexpensive to produce Expensive to produce
Skills required are low Training is required for the technology used
Time scale is short Time scale is long for hybridomas
Produces large amounts of non-specific
antibodies
Can produce large amounts of specific antibodies
Recognizes multiple epitopes on any one antigen Recognizes only one epitope on an antigen
Can have batch-to-batch variability Once a hybridoma is made, it is a constant and
renewable source

26.  What is “Genomic Library”
 - A genomic library is a collection of the total genomic
DNA from a single organism.

27.  C-DNA Library (C for Complementary)
 cDNA is created from a mature mRNA from a eukaryotic cell
with the use of an enzyme known as reverse transcriptase.
 Steps:
 1) Isolation of m-RNA from eukaryotic cell.
 2) Convert m-RNA into DNA using Reverse Transcriptase.
 3) Insert it into Bacterial Plasmid.
 4) Insert plasmid into bacteria and grow in multiple copies.
 5) Isolate plasmid and purify DNA.
 The enzyme Reverse Transcriptase (RT) plays a central role in the
transmission of a broad variety of genetic elements . The enzyme is
responsible for the transcription of viral RNA to produce a double stranded
DNA (dsDNA) that can be inserted into its host genome.

28.  What is “Cosmid”?
 -A cosmid is a type of hybrid plasmid that contains
a Lambda phage cos sequence. Cosmids' (cos sites +
plasmid = cosmids) DNA sequences are originally from
the lambda phage.
 -Enterobacteria phage λ (lambda phage, coliphage
λ) is a bacterial virus, or bacteriophage, that infects the
bacterial species Escherichia coli(E. coli).

29.  What is “BAC” (Bacterial Artificial Chromosome)
 -A bacterial artificial chromosome (BAC) is a DNA
construct, based on a functional fertility plasmid (or F-
plasmid), used for transforming and cloning in bacteria,
usually E. coli.

30.  Shuttle Vectors /Yeast Vectors
 -A shuttle vector is a vector (usually a plasmid) constructed so
that it can propagate in two different host species . Therefore,
DNA inserted into a shuttle vector can be tested or
manipulated in two different cell types.
 Shuttle vectors include plasmids that can propagate
in eukaryotes and prokaryotes (e.g. both Saccharomyces
cerevisiae and Escherichia coli) or in different species of
bacteria (e.g. both E. coli and Rhodococcus erythropolis). There
are also adenovirus shuttle vectors, which can propagate in E.
coli and mammals.
 Shuttle vectors are frequently used to quickly make multiple
copies of the gene in E. coli (amplification). They can also be
used for in vitro experiments and modifications
(e.g. mutagenesis, PCR)
 One of the most common types of shuttle vectors is
the yeast shuttle vector

31.  Electroporation:
 Electroporation or
electropermeabilization, is a
molecular biology technique in
which an electrical field is
applied to cells in order to
increase the permeability of
the cell membrane, allowing
chemicals, drugs, or DNA to be
introduced into the cell.
 Microinjection:
 Microinjection is the use of a
glass micropipette to inject a
liquid substance at a
microscopic or borderline
macroscopic level.

32.  Liposome mediated gene
transfer:
 Liposomes are the sphere of
lipid which can be used to
transport the molecules of
interest into the cell.
 Silicon Carbide whisker
mediated gene transfer:
 In this process the cells are
agitated with silicon carbide,
plasmid DNA.

33.  Calcium Chloride Transformation:
 Calcium chloride (CaCl2) transformation is a laboratory
technique in prokaryotic (bacterial) cell biology. It increases the
ability of a prokaryotic cell to incorporate plasmid DNA allowing
them to be genetically transformed. The addition of calcium
chloride to a cell suspension promotes
the binding of plasmid DNA to lipopolysaccharides (LPS). Positively
charged calcium ions attract both the negatively
charged DNA backbone and the negatively charged groups in the
LPS inner core. The plasmid DNA can then pass into the cell
upon heat shock.
 PEG Transformation:
 PEG helps for easy entry of gene into cell by increasing permeability
through cell wall.

34.  DEAE dextran mediated gene transfer:
 Diethylaminoethyl –Dextran (DEAE-Dextran or
DEAE-D) is use for transfecting animal cells with
foreign DNA. It is added to solution containing DNA
meant for transfection. It binds and interacts with
negatively charged DNA molecules and via a largely
unknown mechanism brings about the uptake
of nucleic acids by the cell.

35.  Colony Hybridization:
 It is a method has been developed whereby a very large
number of colonies of Escherichia coli carrying different
hybrid plasmids can be rapidly screened to determine
which hybrid plasmids contain a specified DNA sequence
or genes.
 Blotting techniques:
 Some techniques like western blot are used to determine
specific cells containing required genes.

36.  HUMULIN
 Insulin (medication): is the use of insulin and similar
proteins as a medication to treat disease like diabetes.
 Humulin was the first medication produced using
modern genetic engineering techniques discovered in
1982 by Eli Lilly.
 The entire procedure is now performed using E.coli,
However, yeast can also be used for this process.
 The daily treatment of millions of diabetic patients
worldwide using Humulin explains the importance of
Recombinant DNA Technology.

37.  Biological implications of genetically engineered
Recombinant human insulin.
 Human insulin is the only animal protein to have been
made in bacteria in such a way that its structure is
absolutely identical to that of the natural molecule. This
reduces the possibility of complications resulting from
antibody production.
 Initially the major difficulty encountered was the
contamination of the final product by the host cells,
increasing the risk of contamination in the fermentation
broth.
 This danger was eradicated by the introduction of
purification processes. When the final insulin product is
subjected to a number of tests, including the finest
radio-immuno assay techniques, no impurities can be
detected.

40.  HUMATROPE
 Humatrope (somatotropin
or somatropin) is a polypeptide
hormone of rDNA origin. Manufactured
by Eli Lilly and Company, it is used to
stimulate linear growth in pediatric
patients who lack adequate
normal human growth hormone. It has
191 amino acid residues and a molecular
weight of 22,125 daltons.
 Its amino acid sequence is identical to
that of human growth hormone of
pituitary origin (anterior lobe).
Humatrope is synthesized in a strain
of E. coli by incorporating a gene for
human growth hormone into plasmid.

41.  ACTIVASE
 Activase (Alteplase) is a tissue plasminogen
activator produced by recombinant DNA
technology. It is a sterile,
purified glycoprotein of 527amino acids.
 It is synthesized using the
complementary DNA (cDNA) for natural
human tissue-type plasminogen activator
obtained from a human melanoma cell line.
 Tissue plasminogen activator is a
protein involved in the breakdown of
blood clots. It is a serine protease found
on endothelial cells, the cells that line
the blood vessels.
 As an enzyme, it catalyzes the conversion of
plasminogen to plasmin, the major enzyme
responsible for clot breakdown helps in
treatment of embolisms.

42.  The manufacturing process involves the
secretion of the enzyme alteplase into the
culture medium by an established
mammalian cell line (Chinese
Hamster Ovary cells) into which the cDNA
for alteplase has been genetically inserted.
Fermentation is carried out in a nutrient
medium containing
the antibiotic gentamicin, 100 mg/L.
 However, the presence of the antibiotic is
not detectable in the final product.
 Phosphoric acid and/or sodium hydroxide
may be used prior to lyophilization or for pH
adjustment.
 Activase (alteplase) is a sterile, white to off-
white, lyophilized powder for intravenous
administration after reconstitution with
Sterile Water for Injection, USP.

43.  Recombinant Human Haemoglobin (r-Hb)
 Hemoglobin is the protein molecule in red blood cells
that carries oxygen from the lungs to the body's
tissues and returns carbon dioxide from the tissues
back to the lungs.
 The continuous development of recombinant
techniques opened the possibility to the production of
Hb in micro-organisms (E.coli).
 Transfusion experiments performed on mice showed
that recombinant Hb maintains physiologically
relevant oxygen and heme affinity.

44.  GENE THERAPY
 Gene therapy is the use of nucleic acid polymers as
a drug to treat disease by therapeutic delivery into a
patient's cells, where they are either expressed as
proteins, interfere with the expression of proteins, or
possibly even correct genetic mutations.
 The most common form of gene therapy involves
using DNA that encodes a functional, therapeutic gene
to replace a mutated gene.
 In gene therapy, the nucleic acid molecule is packaged
within a "vector ", which is used to get the molecule
inside cells within the body.

45.  As of 2014, gene therapy was still generally an experimental
technique, although in 2012 Glybera became the first gene
therapy treatment to be approved for clinical use in
either Europe or the United States after its endorsement by
the European Commission, as a treatment for a disease caused
by a defect in a single gene, lipoprotein lipase (Maintain lipid
level, results in pancreatitis).
 Scientists focused on diseases caused by single-gene defects
LIKE sickle cell anemia.
 In gene therapy, DNA must be administered to the patient, get
to the cells that need repair, enter the cell, and express a
protein in a medically useful way.
 Generally the DNA is incorporated into an
engineered virus that serves as a vector, to get the DNA
through the bloodstream, into cells, and incorporated into
a chromosome.

46.  TYPES OF GENE THERAPY
 It is of two main types-
 1) Somatic gene therapy &
 2) Germline gene therapy
 Somatic Gene Therapy:
 As the name suggests, in somatic gene therapy, the
therapeutic genes are transferred into the somatic cells
(non sex-cells), or body, of a patient.
 It is not inherited by the patient's offspring or later
generations.

47.  Several somatic cell gene transfer experiments are
currently in clinical trials with varied success.
 Over 600 clinical trials utilizing somatic cell therapy are
underway in the United States.
 Most of these trials focus on treating severe genetic
disorders including immunodeficiencies
like haemophilia, thalassaemia, and cystic fibrosis.
 A complete correction of a genetic disorder or the
replacement of multiple genes in somatic cells is not yet
possible. Only a few of the many clinical trials are in the
advanced stages.

48.  GERMLINE GENE THERAPY:
 In germline gene therapy, germ cells (sperm or eggs) are
modified by the introduction of functional genes, which
are integrated into their genomes.
 Germ cells will combine to form a zygote which will divide
to produce all the other cells in an organism and therefore
if a germ cell is genetically modified then all the cells in
the organism will contain the modified gene.
 This would allow the therapy to be heritable and passed
on to later generations.
 Although this should, in theory, be highly effective in
counteracting genetic disorders and hereditary diseases,
some jurisdictions, including Australia, Canada,
Germany, Israel, Switzerland, and the Netherlands……….

49. prohibit this for application in human beings, at least for
the present, for technical and ethical reasons, including
insufficient knowledge about possible risks to future
generations.
 The USA has no federal legislation specifically addressing
human germ-line or somatic genetic modification.

50.  VECTORS USED IN GENE THERAPY
 It has two main methods-
 1) Viral method-
 All viruses bind to their hosts and introduce their genetic
material into the host cell as part of their replication
cycle.
 Therefore this has been recognized as a possible strategy
for gene therapy, by removing the viral DNA and using the
virus as a vehicle to deliver the therapeutic DNA.
 A number of viruses can be used for human gene therapy,
like retrovirus, adenovirus, lentivirus, herpes simplex
virus, vaccinia virus etc.

51.  2) Non-viral Methods-
 Non-viral methods can present certain advantages over
viral methods, such as low host immunogenicity.
 There are several methods for non-viral gene therapy,
including the injection of naked DNA, electroporation,
the gene gun, sonoporation, magnetofection.

52. SOME MAJOR PROBLEMS OF GENE THERAPY
 1) Short-lived nature of gene therapy
 2) Immune response
 3) Difficult to treat multigene disorder
 4) If the DNA is integrated in the wrong place in the
genome, for example in a tumor suppressor gene, it could
induce a tumor.
 5) The cost – only a small number of patients can be
treated with gene therapy because of the extremely high
cost (Alipogene tiparvovec or Glybera, for example, at a
cost of $1.6 million per patient was reported in 2013 to be
the most expensive drug in the world)

53. Transgenic Plants
 A plant which bears a foreign gene of desired function of
other organism is called transgenic plant.
 India's population is expected to reach about 1.5 billion.
It is hoped that 30% India's population will be suffering
from malnutrition.
 These challenges can be met by producing more
nutritious and more productive crops.
 As per estimate made in 2002, transgenic crops are
cultivated world-wide on about 148 million acres (587
million hectares) lands by about 5.5 million farmers.

54.  Steps for making a “Transgenic Plant”
 1) Isolate DNA that codes for the protein you want to
express.
 2) Insert the DNA into a plasmid.
 3) Insert the plasmid into bacteria. Grow a large amount
of bacteria (e.g. Agrobacterium tumefaciens ) containing
this plasmid.
 4) Dip the flowering plant into a large amount of
bacteria & Give bacteria the opportunity to insert the
DNA into the plant cells.
 5) Select for plants that have the insertion.


55. APPLICATIONS OF TRANSGENIC PLANTS
 1) Insect Resistance
 There are a large number of mites, and insects that
attack crop plants and cause great loss in quality and
yield.
 The synthetic insecticides which use to protect from
insects can be a serious threat to the health of plants,
animals and humans.
 The alternative and novel ways of rescue from damages
of insects are the use of transgenic technology. It is eco-
friendly, cost-effective, sustainable and effective way of
insect control.

56.  The cry genes of Bacillus thuringiensis (commonly called
Bt gene) was found to express proteinaceous toxin inside
the bacterial cells.
 When specific insects (species of Lepidoptera, Diptera,
Coleoptera, etc.) ingest the toxin, they are killed.
 The insecticidal toxin of B. thuringiensis has been
classified into the four major classes: cry I, cry II, cry III
and cry IV based on insecticidal activities against many
insects.
 They do not harm the silkworm and butterflies or other
beneficial insects.
 Using biotechnological approaches many transgenic
crops having cry gene i.e. Bt-genes have been developed
and commercialized.

57.  Some examples of Bt- crops are brinjal, cauliflower,
cabbage, corn, cotton etc.
 In India, Bt-cotton was permitted to sow at large scale in
field. It contains crylA (c) gene that provides resistance
against bollworm (Helicoperpa armigera) which is crylA
a notorious pest of cotton.
 2) Virus Resistance
 Plant viruses causes severe disease on crop plants and
result in yield loss in several economically important
plants.
 There are two approaches for developing genetically
engineered resistance in plants: pathogen-derived
resistance (PDR) and non-pathogen-derived resistance
(non-PDR).

58.  In these methods, complete or part of viral gene is
introduced into the plant which interferes the essential
steps in the life cycle of the virus.
 For the first time Roger Beachy and co-workers
introduced coat protein (CP) gene of tobacco mosaic
virus (TMV) into the tobacco. They observed the
development of TMV-resistance in transgenic plants.

59.  Resistance against Fungi & Bacteria
 The fungal and bacterial pathogens attack host plants.
There occurs plant-pathogen interactions. Consequently
plants respond through several defence responses such as
pathogenesis-related proteins (PR proteins e.g.
Chitinase).
 After introducing desired genes into plants several fungal
and bacterial transgenic plants have been produced.
Some pathogen-resistant plants have been
commercialized.
 In 1991, Broglie and co-workers expressed bean chitinase
gene in tobacco and Brassica napus. Such transgenic
plants showed enhanced resistance to a fungal pathogen
Rhizoctonia solani.

60. Other Applications
 Stress Tolerance
 During normal growth of crop number of environmental
stresses may affect including drought, effect of
herbicides etc.
 In such cases it is necessary to introduce the anti-stress
genes so that plant can withstand against these stress
conditions.
 Production of Drought Resistance
 Tested researches have found that there are some genes
present in plant that can protect plant from dehydration
i.e. water loss.

61.  The researchers at the International Maize and Wheat
Improvement Centre have initially focused on
incorporating a type of DREB gene (encoding a
dehydration-responsive element binding protein), which
enables the wheat plants to withstand extreme water
loss.
 Unfortunately, when this gene is continually switched
on, plants are smaller and produce much lower yields
than unmodified varieties. This causes a significant
disadvantage when it comes to plant breeding.
 But the scientists then found that by fusing the DREB
gene with the promoter region of another gene (rd29A),
it is switched on only under the stress conditions of
dehydration or cold temperatures. This results in a
normal growth pattern and yield of plants in good
conditions.

62.  Delayed Fruit Ripening
 A major problem in fruit marketing is the pre-mature
ripening and softening during transport of fruits.
 During ripening genes encode the enzyme cellulase and
polygalacturonase.
 Therefore, ripening process can be delayed by
interfering the expression of these genes.
 In the U.S.A. a transgenic tomato named FlavrSavr
(flavour saver) was produced where ripening is delayed
by lowering polygalacturonase activity.
 A plant growth hormone ethylene is produced during
fruit ripening and senescence. It is synthesized from S-
adenosylmethionine through an intermediate
compound 1-aminocyclopropane- 1-carboxylic acid
(ACC). There is a large number of bacteria that can
degrade ACC.

63.  Therefore, bacterial gene (for ACC) deaminase
associated with ACC degradation was isolated and
introduced into tomato. In transgenic tomato fruit
ripening was delayed because it synthesized lower
amount of ethylene (due to inhibition in ACC synthesis)
than the normal tomatoes.

64.  Transgenic Plant as a “Bioreactor”
 In recent years transgenic plants are used by
biotechnology industries as 'bioreactor' for
manufacturing special chemicals and pharmaceutical
compounds.
 In successful trials transgenic plants have been found to
produce monoclonal antibodies, proteins, vitamins and
the polymer polyhydroxybutyrate (PHB). The PBH can be
used to prepare biodegradable plastics.
 Transgenic plants can be used for the preparation of
edible vaccines, in such process antigenic substances are
introduced in the edible plant which when ingested
release antigen in the blood against which antibodies can
be produced.