1. Recombinant DNA technology involves cutting genes from donor DNA, inserting them into a vector plasmid, and introducing the recombinant DNA into a host cell. 2. There are three main methods of gene transfer between organisms - transformation, transduction, and transfection. Transformation involves uptake of naked DNA by bacterial cells. 3. Vectors like plasmids are used to carry foreign DNA fragments into host cells. Plasmids are small, self-replicating DNA molecules naturally found in bacteria.

1. 1
BIOTECHNOLOGY: Process and Applications
Methods of gene transfer:
The vector containing DNA or genes must be transferred into living cells
then desirable gene can be multiplied and expressed. It can be done by following
methods,
A Transformation
B Transduction
C Transfection
A. Transformation- It is the process by which a cell pick up naked DNA
segment or vector DNA from surroundings and attached with own Chromosomal
DNA. It is the method of introducing desired or foreign DNA into bacterial cell.
Eg. E.Coli .
B. Transduction: It is the process of transfer of DNA from one organism to
another through Bacteriophage (type of virus). The bacteriphage viruses can
naturally infect the cells and inject the DNA into host cell. This is known as
transduction.
C Transfection: It is the method by which transfer of foreign DNA into
eukaryotic cells, like plant and animals.
Process or Technique of Recombinant DNA Technology:
Recombinant DNA technology is involves cutting and pasting of DNA
fragments in the vector plasmids.
It involves following steps-
1. Isolation of desired gene
2. Selection of suitable vector
3. Construction of recombinant DNA
4. Introduction of r DNA in to host cell
5. Selection and multiplication host cell containing r.DNA
molecule

2. 2
1. Isolation of desired gene:
1. The cells having desired gene are cultured.
2. The desired gene is removed from selected cells by lysing with using
suitable enzyme.
3. The isolated DNA is ultracentrifugated and purified.
4. Now isolated DNA is called DONOR DNA.
2. Selection of suitable vector:
1. Desired DNA is then inserted into vector DNA, which may be
plasmid, phage DNA, cosmids, YAC ( Yeast Artificial Chromosome).
3. Construction of recombinant DNA:
1. Both the donorDNA and vector DNA are cut at both ends by same
restriction enzyme so that they have same complementary sticky or
cohesive ends.
2. Then isolated DNA is inserted into cut open vector DNA.
3. The complementary sticky ends of both the DNA are joined with
DNA ligase enzyme.
4. Then this plasmid DNA is called as recombinant DNA or Chimeric
DNA.
5. The process ofcutting and joining of desired gene with vector DNA is
called splicing.
4. Introduction of r DNA into host cell:
1. The r DNA is then transferred into host cell. eg. E. coli
2. The host cell should be without plasmid, non pathogenic and
harmless.
3. The transfer of r DNA into host cell may take place by
transformation or transduction or vectorless gene transfer.

3. 3
4. Commonly, by means of electroporation in which brief
impulses of high voltage of electricity are applied to protoplast
suspension containing naked recombinanat DNA.
5. Electroporation create pores on cell membrane and make them
permeable for uptake of r DNA.
6. Host cell may take up naked r DNA from medium under
suitable condition.
7. The bacterial cell containing r DNA is called transformed cell.
8. The r DNA integrate with host DNA express to produce new
character in the cell.
5. Selection and multiplication host cell containing r.DNA molecule:
1. The cells with r DNA are selected and allowed to divide.
2. It results in formation of multiple copies of desired gene called
cloning.
3. These cells have a copy of r DNA.
4. Cloning can be done artificially by PCR method or by
amplification.
5. Then the cells with r DNA are screened and ready to transfer
into recipient organisms.

4. 4
Applications of r DNA:
1. Many microorganisms are genetically modified to produce many
useful chemicals like, insulin, human growth hormone, interferon,
vaccines, anticoagulants etc.
2. To synthesize the desirable gene artificially.
3. It helps to cure many genetic disorders like hemophilia by gene
therapy.

5. 5
4. To produce plant varieties with desirable characters.
5. To produce transgenic plants which are resistant to fungal diseases
and pest.
6. To diagnose and care the genetic diseases by using recombinant
DNA technology.
Following are the some products of r DNA technology:
Groups R DNA products Applications
Blood proteins 1.Erythropreteins
2.Blood clotting factors
VII, VIII, IX
3.Tissue plasminogen
activator
4.Urokinase
Anemia and kidney
disease, AIDS.
Replacement of
clotting missing in
patients of hemophilia
A and B
Dissolving blood
clots after heart attack
and stroke.
Blood clots
Human
Hormones
1 Epidermal growth factor
2Folicle stimulating
hormone (FSH)
3Insulin (Humulin)
4 Nerve growth factor
Relaxin
5 Somatotropin
Burns
Treatment of diabetes
mellitus
Nerve damage
Delivery
Growth defects
Immune
modulators
1 Interferon (αβγ)
2Colony stimulating factor
3 tumor necrosis factor
4 Lysozyme
Treatment of
pathogenic viral
infections
cancer multiple
sclerosis
Cancer
Cancer

6. 6
Vaccines 1Hepatitis B
2 Measles vaccine
3 Rabies vaccine
4 Cytotmegalo virus
Prevention of
infectious diseases
like Hepatititis B,
Herpes measles,
Rubella etc
Transposons or Jumping Gene: The some nucleotides sequences of
DNA strands move from their original position to other position of same
DNA strands or chromosome are known as transposons or jumping
genes.
OR
Transposons are some repetitive DNA sequences can change their
location in DNA or genome.
The first transposon was discovered by Barbara McClintok (1951) in
Maize (Zea mays). She was awarded Nobel Prize of 1983.
1. Transposons may associate with some gene.
2. Transposons are mobile and can move to any place in target
chromosome.
3. They may undergo mutation and modify the action.
4. They possess repetitive nucleotide sequences and 5-7 nucleotides
long.
5. These are the DNA segments which can undergo excision or
insertion from and into different locations of bacterial chromosome
or DNA of plasmids or viruses.
Transposition: The phenomenon of movement of transposons or
specific pieces of DNA in the genome is called transposition.

7. 7
Types of Transposons: On the basis of their mechanism of
transposition, the transposons are two types as,
a. Retrotransposons (class I)
b. DNA transposons (class II)
a. Retrotransposons (class I):
1. These transposons copy in two stages, first from DNA to RNA and
then from RNA back to DNA by reverse transcription.
2. The copy of RNA is also called as retrotransposon.
3. Initially a copy of RNA is formed from a DNA by normal process
of transcription.
4. Then by using enzyme reverse transcriptase, DNA is copied from
RNA.
5. The newly formed DNA is the copy of the transposon inserted in
the new position or location.

8. 8
6. As a result retrotransposition, there are now two copies of
transposon at different locations on the genome or chromosome.
7. Thus transposition is nothing but cut and paste mechanism.
b. DNA transposons (class II):
1. DNA transposons are able to direct transposition or movement
from DNA to DNA.
2. They do not involve RNA intermediate.
3. These transposons require enzymes which are coded by the genes
within transposons.
4. These enzymes are called transposae enzyme.
5. The transposae enzymes make cut at the target site to produce
sticky ends.
6. This cut the transposon and reintegrates it at new site with the help
of DNA ligase.
Eg. 1. In case of Maize, a segment of DNA moved into gene coding
for pigmented kernels and produced light coloured kernels. It was
found that nearly 50% of the total genome of Maize consists of
transposon.
2. In human the most common type of transposons belongs to Alu
family having a site for cutting by restriction enzymes Alu I. The
number of nucleotides or bases per transposon is about 300. It has
300000 copies in the genome.
Significance of Transposons:
1. They alter the cells genome size.
2. They bring about reshuffling nucleotide sequence in gene or
DNA.
3. They can create phenotypic changes( external features)
4. Exon shuffling leads to develop new genes.
Plasmids (5-10): are small extra chromosomal, double stranded
circular self replicating DNA or mini chromosome.

9. 9
The term plasmid was introduced by the American molecular
biologist, Joshu Lederber (1952).
Occurrence:
1. plasmids is naturally found in the cytoplasm of bacterial cells
2. They are rarely found in yeast, plant and animal cells.
3. The number of plasmids 10 to 100 per cell.
Characters of plasmids:
1. It is capable of self replication.
2. The most of the plasmids are required for survival of the host
in which they live.
3. They carry genes which control some of the metabolic
activity which are essential to survive and reproduction.
4. The size of plasmid varies from 1 kb to 1000 kb.
5. Plasmid cannot accommodate long DNA fragment hence
cosmids are constructed.
6. Plasmids are easily isolated from bacterial cells because of
not a part of main chromosome.
7. Plasmids are used cloning vehicle, to carry the donor DNA.
8. They retain their properties even after combining their DNA
with the DNA of other organisms.
9. Ex. pUC, pBR 322 plasmid of E.coli.
10. The Ti plasmid of Agrobcterium tumefacens a soil
bacterium used for gene transfer in plans.
11. The most widely used versatile, manipulated vector is
pBR 322 an ideal plasmid vector.
Fig: Bacterial Cell with Plasmids

10. 10
Fig: Structure of Plasmids

11. 11
Fig: Structure of pBR 322 Plasmid

12. 12
Fig. Plasmid Structure

13. 13
Nomenclature Of plasmids:
Plasmids are named as plasmid is denoted by small letter p. followed by
first letter of researcher (s) names and the numerical number given by
the inventers.
Nomenclature of pBR 322 plasmid:
This was the first artificial cloning vector prepared in 1977 Boliver and
Rodriguze.
*Here p denotes plasmids, BR – the name of the scientists, Boliver and
Rodriguze, 322- number given to designate this plasmid.
Some plasmids are given the names of the places where they were
discovered. For eg. pUC, p- plasmid, UC- it was discovered in
University of California.
Cosmids: are the plasmids having a DNA fragment of bacteriophages λ,
virus. It possesses characters of both plasmid and bacteriophages.
Plasmids are unable to carry large fragments of foreign DNA. They are
constructed to carry large fragments of DNA up to 45 kbp.
Bacteriophages (10-15 kb) (Phagein- to eat):
These are the viruses that infect to bacterial cells and kill bacteria.
In short they are called phages.
These vectors are used for cloning large DNA fragments for the
construction of gene bank or gene libraries. The most commonly used
phages are λ phages and M13 which infect E.coli to understand how
they work as cloning vectors.
Structure of Bacteriophages (10-15 kb):
The infectious units of virus are called viron.
Structurally each phage consists following parts
1.Head is covered by Capsid, a protein coat

14. 14
2. Central core of nucleic acid
3. Tail
4. Foot
5. Tail fibers and pegs
6. Genetic material may consist of DNA or RNA.
7. Capsid protects the nucleic acid core against the action of nuclease
enzyme.
8. DNA molecule may be cyclic or linear.
9 With respects number of DNA strands, 4 types of nucleic have found
as double stranded (ds DNA), single stranded (ss DNA), double stranded
RNA (ds RNA) and single stranded RNA (ss RNA).
10. Tail of phage tube is connected with proteinaceous tail sheath.
11. The sheath is connected to a thin disc like structure called collar at
upper end and base plate at the lower end.
12. The base plate is hexagonal and has a pin or spike at each corner.
13. From each corner thin long tail fibers are produced, which help to
attachment of host cells.
14. The large DNA molecules can be injected in host bacterial cell by
bacteriophages.
15. The most commonly used bacteriophages as cloning vectors are M13
and lambda phage.
14. The virulent portion of phage DNA is removed and desired DNA is
loaded and retained in phage capsid.
Size: size of viruses is variable, observed under electron microscopy and
centrifugation etc.

15. 15
Shape: occure in three main shape spherical (polyhedral), helical
(cylindrical or rod like) and complex (tadpole like).
Fig: Structure of Bacteriophages (Virus)

16. 16
Phage lambda (λ) as vector:
1. Phage lambda contains a proteinaceous head and long
tail attached to head.
2. DNA of lambda phage is linear and, double stranded
48.5 kbp long.
3. DNA is with cos sites of 12 bases at ends.
4. The most important feature of lambda genome is that
half of genome (only 50-60%) required for a lytic
growth of phage i.e. for its multiplication and function.
5. 40-50% or half of phage genome can be replaced
foreign DNA.
6. The portion of phage DNA required for packing it in
capsid is retained and some of the nonessential,
pathogenic part of genome can be removed.
7. The phage DNA has 12 bases at each end is paired but
complementary.
8. These ends are sticky or cohesive and as cos sites.
9. These cos sites are important for insertion of DNA into
phage head.
10. Lambda DNA remains linear in the phage head.
11. But within host cells the two cohesive end join to
each other to form circular DNA molecule.
12. Circular DNA is must for replication.
13. Lambda vectors allow cloning of DNA segments
up to 20-25 kbp length.
14. The rDNA is packed with in viral particles in vitro
and allowed to infect host cell i.e. E.coli which have
been cultured on agar medium.
15. Within host cell rDNA starts multiply and after
completion and formation of multiple copies of phages
the bacterial cells burst to releases phages. It called lytic
cycle.

17. 17
Viral life Cycle:
The bacteriophages infect to bacterial cell and multiply within the
host cell. It is called as viral life cycle.
The bacteriophages multiply by two ways,
1. The Lytic cycle and 2. The lysogenic cycle
1. The Lytic cycle:
Some viruses multiply as soon as they enter into host cell and it
ends with breakdown of host cell. Such viruses are called lytic viruses
and the mode of infection is virulent. It cause the death of host cell.
2. The Lysogenic cycle:
In this type, the viruses do cause lysis or break down of host cells
but host cell remain live. Such viruses are called lysogenic viruses and
the mode of infection is temperate. Eg. lambda phage of bacterium
E.coli.
The lytic cycle is complete in following steps as,
1. Attachment
2. Penetration
3. Synthesis of proteins and nucleic acids
4. Viron assembly or maturation
5. Lysis and release of phages
1. Attachment:
1. When phage particles colloids on host cells and get attached
to its surface.
2. As phage particles do not move independently.
3. They depend upon the random and unexpected meetings with the right
receptors.

18. 18
2. Penetration:
1. After the attachment tail fibers of phages bring the base plate
closer to the surface of host cell.
2. The enzymes produced by phage plate digest host cell wall
and to form the pores.
3. Through pores phage injects its DNA into the host cell.
4. The capsid and tail remains outside and it is called ghost.
3. Synthesis of proteins and nucleic acids:
1. Phage disturbs host normal synthesis of proteins and nucleic
acids.
2. It forced to synthesis of viral DNA and proteins.
3. As a result new viral particles are produced.
4. Viron assembly or maturation:
1. First the base plates are gets assembled with the tail.
2. The heads and capsid are constructed separately and then
joined to tails.
3. The DNA is always getting packed within the capsid of
heads.
4. The whole process complete within 15 minutes.
5. Lysis and release of phages:
1. Within 15 minutes large number of phages is produced after
the entry of phage DNA into host cell.
2. The host cells burst to releases phages it is called lysis of cell
3. The lysis of cells is done by enzyme endolysin which breaks
the host cell wall.
4. Then the released phages are ready to infect new host cells.

19. 19

20. 20
Fig. Lytic Cycle
Restriction Fragments: is a DNA fragment produced by the
cutting of DNA strand due the action of specific restriction
endonucleases enzyme.
Restriction: The process of cutting of DNA strands at specific site
is called restriction.
Steward Linn and Werner Arber (1963) isolated two enzymes
which restricted growth of bacteriophages in E. coli bacterium.

21. 21
One of the enzymes which cut the DNA strands called
endonuclease. Restriction enzymes belong to the class Nucleases.
Types of Nucleases: two types as follows,
1. Exonucleases- this type of nucleases remove the
nucleotides from the end of DNA strands.
2. Endonucleases- this type of enzyme cuts at specific
positions within DNA strand.
Restriction endonucleases commonly used as molecular scissors or
chemical knives or scalpels.
Types of Restriction endonucleases:
There are three main types of REN as
1. Type I, 2. Type II and Type III
Out of three only Type II RENs are used in r DNA technology.
Type II enzymes are simple and can be used in Vitro.
They identify and can cut (cleave) the specific DNA sequences up
to 4-8 nucleotides base pairs.
Today more than 350 different type II endonucleases with 100
recognition sites are known.
Nomenclature of REN:
1. REN are named by standard procedure.
2. They are named by bacterium from which they have been
isolated.
3. The first letter used in capital and in italic from the first letter
of the bacterium Genus name.
4. Then two letters in italic come from first two letters of
species name of bacterium.
5. The fourth letter of enzyme name is the first letter of bacterial
strain.

22. 22
6. The fourth letter of enzyme name should be in capital letter.
7. The name end with roman number in which the enzyme was
discovered or isolated.
8. Examples: EcoR I the E from Escherichia, co from coli RY
strain, I first endonuclease to be discovered.
9. Hind III is isolated from Haemophilus (H), inflouenzae (in)
strain RD (d) and the third endonuclease (III) to be
discovered.
10. Hae III- is isolated from Haemophilus (H), aegyptius
(ae) and third endonuclease to be discovered.
Restriction Sequences: is the site where the DNA strand is cut by
restriction endonuclease.
REN have capacity to recognize the specific sequence on DNA strand
and cut each strand at that site.
Each restriction endonuclease is highly specific to recognize specific
DNA sequences.
Restriction site is the sequence of about 4-8 nucleotides.
REN cut both the strands of DNA at the same time.
They cut internal phosphodiester bonds at those specific sites.
The most of restriction sites are palindrome types.
Palindrome Sequences: palindromes are groups of letters that from the
same word when both read forward and backward.
For example, MALAYALAM, MADAM, NITIN.
Palindromes are the mirror images of each other.
This special type of sequences in the DNA recognized by REN is called
Palindromic nucleotides.
The following DNA sequence is read the same on the two strands in 5҆
-3҆҆ direction and can be read same in 3҆-5҆ direction also.

23. 23
→→→→→
5҆-A-C-C-G-A-A-T-T-C-G-C-A-3҆
│ │ │ │ ││││││││││ │
3҆-T-G-G-C-T-T-A-A-G-C-G-T-5҆
←←←←←←
Cleavage Pattern:
1. Sticky Ends or staggered ends or cohesive ends
2. Blunt Ends
1. Sticky Ends or staggered ends or cohesive ends:
1. Restriction endonuclease cut the DNA strand little away from
centre of the palindrome sequences but between the same bases of
the opposite strands.
2. This leaves single unpaired bases at cut ends.
3. These ends with unpaired bases are called sticky ends.
4. These unpaired bases of sticky ends easily paired with
complementary bases of other fragments of DNA.
5. These sticky ends are mostly useful in r DNA technology.
6. Sticky ends are easily joined by ligases.
7. Example – enzyme E.co R I, Hind II Hae III produce (sticky ends).

24. 24
Sticky Ends (unpaired base)
↓
5҆-G A-A-T-T-C-3҆
3҆-C-T-T-A-A G-5҆
↑
Sticky Ends (unpaired base)

25. 25
In agarose gel electrophoresis, restriction fragments develop band
pattern the characteristics of original DNA molecule and restriction
enzyme used.
Relatively small DNA molecules of viruses and plasmids can be
identified simply by their restriction pattern.
Fig. Electrophoresis Apparatus Fig. Agarose Gel showing DNA bands
Gene Library: The collection of cloned DNA fragments that represents
the entire genome of a species is called as gene library.
Types of gene library-
Depending upon the availability of source of DNA, gene library is of
following two types

26. 26
1. Genomic Library: The collection of DNA fragments or genes from a
particular species is called as genomic library.
Construction of Genomic library:
Following steps as
1. Isolation of genomic DNA
2. Cutting of DNA fragments of suitable size
3. Cloning into suitable vector
4. Screening, identification and characterization of clones
1. Isolation of genomic DNA: The cells of source organisms
are grown in tissue culture and complete genome or
chromosomes are isolated from cell (DNA extract).
2. Cutting of DNA fragments of suitable size-
The isolated genome is cut into fragments of suitable size by
using suitable restriction endonucleases.
It forms sticky ends which are compatible with vector.
3. Cloning into suitable vector:
a.The each fragment is joined with vector DNA to form
recombinant DNA.
b. vector DNA may be plasmid or cosmid or bacteriophages.
c. The rDNA is introduced into suitable host cell.
d. Then host cells are cultured on suitable medium.
e. rDNA is multiplied along with the host cells.
f. This produces many copies of genome is called clones.
g. All these clones are stored in suitable medium.
h. Thus all these clones of one genome of same species is
known as genome library.
4. Screening, identification and characterization of clones:
The desired gene of fragment are identified and stored
separately.

27. 27
2. cDNA Library:
1. cDNA is nothing but complementary DNA produced by reverse
transcription from mRNA.
2. cDNA produced by this method is known as Teminism (named
after discoverer Temin and Baltimore).
3. cDNA library is used for eukaryotic organisms.
4. In eukaryotes genes with coding and non coding sequences.
5. Mostly coding sequences of gene undergo transcriptions and
translations to produce specific proteins through mRNA.
6. The mRNA of different organisms at different stages are isolated
and used for production of cDNA with the help of reverse
transcriptase enzyme.
7. cDNA library having different types cDNA with different types of
structural or functional proteins.
8. These cDNA fragments are introduced into suitable vector and
cloning in a particular host cell like E.coli.
9. The products like human insulin, interferons, and blood clotting
VIII factors are produced by cDNA.
Gene Amplification (PCR): it is nothing but to obtain multiple
copies of desired gene or known sequences.
Gene amplification can be done artificially by using polymerase
chain reaction (PCR).
PCR STEPS: it is an artificial in vitro technique of production of
multiple copies of desirable gene.
It was first developed in 1983 by Kary Mullis and awarded Nobel
Prize of chemistry in 1993.
Basic requirement for PCR technique:
1. DNA segment to be amplified (100-35000 bp length)
2. Primers (forward and reverse) synthetic oligonucleotides of
17-30 nucleotides. These are complementary to the sequences
present on the desired DNA segment.

28. 28
3. 4 types of deoxyribonucleotides(dATP, dCTP,
dGTP.dTTP),these are collectively called dNTPs.
4. Thermostable DNA polymerase enzyme that can withstand
upto 94 0C. i.e. Taq polymerase obtained from Thermus
aquatics.
PCR is thermal cycling and alternately heating and cooling
the PCR sample.
Steps of PCR technique: following are the 3 steps,
1. Heat denaturation
2. Annealing
3. Polymerisaton
1. Heat denaturation- heating of DNA at 91 0c.
It breaks the hydrogen bonds to make ssDNA.
DNA molecule with mor G-C pairs need higher temperature.
2. Annealing- is pairing of primers to the ssDNA segment.
The primers are designed as per the requirement.
This step require temperature at about 55 0C.
3. Polymerisation- The temp. is increased up to 72 0c.
The Taq Polymerase adds dNTPS behind the primers on the
ssDNA.
These 3 steps are collectively called as one cycle.
This process is carried out for about 28-30 cycles.
Each cycle takes about 3-5 minutes.
The DNA produced is used as template for replication.
PCR is carried out in automotive machine.

29. 29
Application of PCR technique:
1. Used in DNA cloning.
2. Gene amplifications.
3. DNA based polygeny or functional nalysis of gene.
4. Diagnosis of hereditary diseases.

30. 30
5. DNA fingerprinting to solve the parental disputes, crimes etc,
6. Diagnosis of infectious diseases.
7. Diagnosis of cancer.
Application of Biotechnology in Agriculture- Bt Crops:
Today Biotechnology is nothing but revolutionized research activities in
the field of agriculture.
To increase the yield of crop or food production following things are to
be practiced
1. Use of chemical fertilizers and pesticides.
2. Use of bio-fertilizers and bio-pesticides.

31. 31
3. And use of genetically modified crop plants or transgenic plants or
Bt.plants.
Transgenic plants with desirable characters such as disease, insect pest
and herbicide resistance can be produced.
Plants with better photosynthetic efficiency, nitrogen fixing ability,
improved storage proteins, and higher vitamin content can be produced.
The most of current commercial application of modern biotechnology in
agriculture aim to reducing the dependence of farmers on agrochemicals.
Therefore inset resistant crops or Bt crops are produced.
Bacillus thuringiensis :
1. It is soil bacterium.
2. It produces protein with insecticidal property, to kill insects.
3. Today the spray produced from this bacterium is used on crop
plants.
4. It produces some types of protein crystals i.e. thurocide.
5. Thurocide has toxin property to kill insects.
6. It nothing but a Bt toxin.
7. Thurocide is exists in inactive protoxins.
8. When it inject into insect body it become active form of toxin due
to alkaline PH of the insect gut or alimentary canal.
9. Due to alkaline PH thurocide absorb water and swell up to bursts
the insect alimentary canal to cause the death of the insect.
10. Now days the Bt gene is transformed into plants by rDNA
technique.
11. The Bt gene is called CRY gene, it express to produces
protoxins when insect ingests the transgenic plants and insects
killed.
12. Bt cotton is commercially available in market to control the
disease affecting the cotton bolls (boll worm).
13. Today Bt gene is cloned and introduced in many plants to
control the insects, eg. Bt corn, rice, tomato, potato and soybean,
etc.

32. 32
Agrobacterium tumefaciens:
1. It is a free living soil bacterium.
2. These bacteria infect to plants and causes crown gall disease to
host plants.
3. Agrobacterium is having plasmid with tumor inducing gene
called Ti DNA (Ti plasmid).
4. Ti DNA is mainly responsible to produce tumor in host plant
cell.
5. Into this Ti DNA the desired gene is introduced by replacing
non essential gene.
6. The tumor inducing gene of T DNA is nothing but the marker
gene.
7. Through Ti plasmid the desired gene like Nif gene (nitrogen
fixing gene) is introduced in non leguminous plants.
8. Therfore Agrobacterium tumefaciens is commonly used for
gene transfer in plants.
9. Nif gene or Bt gene is cloned inside the A. tumefaciens and then
transferred into desired plants.
10. Many Bt crops transgenic plants or genetically modified
plants (GM plants) are produced using A. tumefaciens.
11. Examples of GM plants, Flavor savor tomato, Golden rice,
etc.
Flavor Savor Tomato: it developed by introducing
polygalacturonase gene into tomato plant.
Polygalacturonase encoding gene antisense to increases longer
shelf life of tomatoes by producing less pectin degrading enzyme
polygalacturonase enzyme.
The flavour of tomatoes is saved which is a additional advantage.
Golden Rice: It is genetically modified rice rich in pro vitamin A,
beta carotene.

33. 33
Bio-safety Issues:
1. Bioethics is nothing but a set of standards which can be used to
regulate our activities in relation to the entire biological world.
2. Biotechnology inventions in the area of agriculture,
pharmaceuticals and health care have raised many ethical issues
recently.
3. The use animals in biotechnology are a type of cruelty, as they
undergo great physical suffering while performing experiments on
them.
4. To obtain various pharmaceutical products like proteins, they are
treated as factors.
5. The transfer of gene from one species to other is violation of
integrity of species.
6. Introduction of human genes to other animals or vice-versa dilutes
the concept of humanness.
7. Biotechnology involves over exploitation of other living beings for
the benefits of mankind.
8. It adversely affects biodiversity and environment.
9. Genetic modification of organisms can have unpredictable effects
when such organisms are introduced into ecosystem.

34. 34
10. Cross pollination between GM plants and wild plants will
lead to contamination of gene pools of wild varieties.
11. Consumption of GM food may develop allergies.
12. GM microbes escape from laboratory and will be hazardous.
13. Therefore manipulations of living organisms need regulation.
14. The Indian government has set up Genetic Engineering
Approval Committee (GEAC).
15. GEAC will decide the validity of GM research and safety of
introducing GM product for public service.
Bio- Piracy: The illegal exploitation of biological resources of other
nation without prior authorization from concern country is called bio-
piracy. OR bio-piracy is nothing but robbery or theft of bio resources.
1. Under developed countries are rich in bio diversity or bio resource
and traditional knowledge while developed countries rich finance
and technology.
2. Developed countries since past two decades have been enjoying
immense profits by patenting the knowledge and bio resources of
underdeveloped countries.
Bio patent: is patent granted by the government to inventor for
biological entities, processes and products.
1. Patent gives owner exclusive rights to use resources, process or
market the product and earn profits.
2. Basmati rice known for its unique aroma (scent) and flavor has
been grown in India for centuries.
3. There are about 27 documented varieties of basmati grown in
India.
4. A Texas based company got patent rights on basmati rice through
US patent and Trademark office.
5. This allowed the company to sell a new variety of basmati Texmati
in the US and abroad.
6. Actual Texmati is obtained by crossing between basmati and semi
dwarf variety and claimed as new invention or new variety.
7. Thus it is the case of bio piracy and unfair bio patenting.

35. 35
8. Patents of turmeric and margosa (Neem) and many other Indian
traditionally known medicinal plants have been pirated by USA.
9. Now we are fighting to cancel the illegal bio patents.
10. We have own the legal battle (rights) against the patents of
Basmati and turmeric.
It is the need for launching a genetic literacy movement
in schools and colleges on the rapid developments taking place
in the field of molecular genetics.
It would helps in better understanding of the opportunities
and risks associated with recombinant technology. It helps to promote
the safe and responsible use of the tools of new genetics in the fields of
food, agriculture, medicine, industry and environment.
--------------------------------------XXX-------------------------------------------