Biotechnology MohanBio

• The application of technology to improve a
biological organism.
natural variation: Allelic differences at genes
control a specific trait.
• Gene - a piece of DNA that controls the
expression of a trait.
• Allele - the alternate forms of a gene.

• Central Dogma of Molecular Genetics
DNA

RNA

Transcription
Translation
Protein
trait or phenotype.
• The application of the technology to modify the
biological function of an organism by adding
genes from another organism.

Biotechnology.
• Utilization of biological entities
and their component in
production of some products for
human welfare is called
biotechnology.
• The contribution of biotechnology
in different field of biology are,

Medical Biotechnology:
• Production of human insulin using recombinant
DNA technology.
• Production of anti biotic like penicillin
erythromycin. Etc.
• Production of mono clonal antibodies using
hybridoma technology.
• Treating defective gene using gene therapy.
• Identification of immigrants. Criminals, disputed
parents, missing baby etc. using DNA finger
printing technology.

Environmental biotechnology.
• Some microbes are used to treat sewage
waste in water purification.
• Detoxification of industrial waste are done
using microbes.
• Some microbes are used to reduce the
percentage of oxides of sulphur in industrial
effluents.
• Degradation of petroleum products and
management of oil spills are done by using
microbes.

Industrial biotechnology.
• Production of useful organic compounds like
ethyl alcohol, lactic acid, citric acid etc. by using
microbes.
• Production of enzymes like amylase, lipase,
protease from microbes.
• Production of bio fuel like ethanol, bio gas etc.
• extraction of some minerals like copper,
uranium, from low grade ore using microbes.

Plant biotechnology or agricultural
biotechnology.
• Rapid multiplication of crop plants, medicinal
plants, forest plants and endangered plants
using tissue culture.
• Production of viral and other pathogen
resistance plants.
• Production of haploid or polyploidy crop
plants to increase yield.
• Production of transgenic plants as nitrogen
fixing plants, insect resistance plants etc.

Animal biotechnology
• To develop Genetically modified animals or transgenic
animals.
• Transgenic cows – increase milk supply and meat
• Transgenic Chickens – more resistant to infections.
• Transgenic Goats, sheep and pigs – produce human
proteins in their milk
• To increase the heard of specific breed using invitro
fertilization and embryo transfer.
• Cloning of animals.
• Transgenic mice – used to study human immune system
• NTBT: National biotechnology board.
• DBT: department of biotechnology.

• Application of biotechnology varies from agriculture to
industry - food, pharmaceutical, chemical, bio-products,
textiles, medicine, nutrition, environmental conservation,
animal sciences etc.
• Admission to the integrated five year M.Tech program
offered by IIT Delhi and Kharagpur is through the Joint
Entrance Exam (JEE).
• Jawaharlal Nehru University, New Delhi conducts a
combined all India level entrance examination for MSc
Biotechnology program.
• Candidates with Bachelor's degree under 10+2+3 pattern
of education in Physical, Biological, Agricultural, Veterinary
& Fishery Sciences, Pharmacy, Engineering, Technology 4Years BS (Physician Assistant Course); OR Medicine (MBBS)
OR BDS with at least 55% marks are eligible to apply for
MSc (Biotechnology) offered by JNU and several other
universities all over the country

A biotechnologist may find jobs in various
quarters. In India Students can mainly explore
job options in the following fields:
• Drug and pharmaceutical research
• Public funded laboratories
• Chemicals
• Environment control
• Waste management
• Energy
• Food processing
• Bio-processing industries

• The government institutes and organizations, such as
Department of Biotechnology (DBT), several
agriculture, dairy and horticulture institutes offer
employment.

• In private sector, Drug companies in biotechnology
like Dabur, Ranbaxy, Hindustan Lever, Dr Reddy's Labs
that have their R & D units offer Biotechnology
professional .
• Even in the food processing industry, chemical
industry and the textile industry.
• The major companies, which hire biotechnologists,
are Hindustan Lever, Thapar Group, Indo American
Hybrid Seeds, Bincon India Ltd., IDPL and Hindustan
Antibiotics etc.

• Institutes Offering B.Tech/M.Tech/PhD :
• Admission to the integrated five year M.Tech
program offered by IIT Delhi and Kharagpur is
through the Joint Entrance Exam (JEE).
• Indian Institute of Technology, Delhi
• Courses Offered: B.Tech., M.Tech. in
Biochemical Engineering and Biotechnology,
M.S. (Research) in Biochemical Engineering and
Biotechnology and Pre Ph.D. Courses
• National Dairy Research Institute, Karnal
• Courses Offered : M.Sc and M.Tech degree in
Animal Biotechnology

• Indian Institute of Technology, kharagpur.
• Courses Offered : B.Tech.(H) in Biotechnology
and Biochemical Engineering, B.Tech.(H) and
M.Tech. in Biotechnology and Biochemical
Engineering, MS (Biotechnology), M.Tech. Biotechnology and Biochemical Engineering,
Ph.D. (Biotechnology)
• All India Institute of Medical Sciences (AIIMS)
• Courses Offered : M Biotech

• Centre for Cellular and Molecular Biology
imparts training to doctoral students in an
academic program linked to the Jawaharlal
Nehru University, New Delhi. Besides, the
Centre also trains post-doctoral fellows though
training programs sponsored by CSIR,
Department of Biotechnology (DBT), and the
Department of Science and Technology (DST),
Govt. of India, New Delhi.
• On an average at any given point of time there
are over 100 such researchers at the CCMB,
including guest workers from various
institutions.

• The students enrolled in academic programs
require to have strong motivation to pursue
research in modern biology leading to a Ph.D
degree.
• The projects offered for Ph.D. cover
specialized areas of Cell Biology, Molecular
Biology, Genetics, Genomics, Developmental
Biology, Nano biology, Plant Molecular
Biology, Membrane Biology, Protein Structure
and Function, Biology
• of Macromolecules, Biology of Infection,
Epigenetics, Chromatin Biology and
Bioinformatics

Genetic Engineering (Gene Manipulation )
• The technique of transferring desired gene to an
organism to manipulate its genome is called genetic
engineering.
Application of Genetic engineering:
• Understanding biological events in biological courses.
• Production of pharmaceutical compounds like
insulin, growth hormone,etc
• Production of transgenic animals.
• Production of transgenic plants.
• Production of pathogen and insect resistance plants.

Tools used in Genetic Engineering.
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Desired gene
Vector
Enzymes: REN, DNA ligase.
Host cell.
Bio reactor.

• Desired gene: The functional or normal gene of
our interest taken from donor cell.
It is also known as foreign gene or trans gene.
• Vector: The carrier DNA that act as vehicle to
carry desired gene to the host cell is called vector.
• The imp vectors used in Genetic engineering are,
1. Plasmid.
2. Phages.
3. Plant virus.
4. Animal virus.
5. Cosmids.
6. Artificial chromosomes.

• Plasmids: The extra chromosomal small circular
self replicating DNA present in bacterial cell is
called plasmid. The number of plasmid varies
from 1 to 20 in a single bacterial cell.

Types of plasmids:
• F+ plasmid: It is the plasmid that contains
fertility factor.
• R plasmid: It is the plasmid that contains
antibiotic resistance gene.
Ex: ampicillin and tetracycline resistance
gene.
• Col plasmid: it is the plasmid that contains col
gene that synthesizes the protein colocin. The
colocin kills the other strains of bacteria.

• Virulence plasmid: It is the plasmid that
contains pathogenic gene.
• Metabolic plasmid: It is the plasmid that
contains gene for metabolic activity.
–Ex: nif + gene.
Common plasmids used in genetic
engineering:
• pBR 322.
• pUC18.
• Ti plasmid. ( tumor inducing plasmid)
• Ri plasmid. ( root inducing plasmid)

pBR 322 plasmid:
• It is the naturally occurring E. coli plasmid.
• It has 4.3 Kbs. (kilo base pair size)
• It contains one Ori site. ( origin of replication
site).
• It contains two antibiotic resistance genes.
Amp+ and Tet +
• It contains specific restriction endonuclease
recognizing site.

pUC 18 plasmid.
• pUC 18 was first constructed at
university of California.
• It has the size of 2.73 kbs
• It contains one ori site.
• The fertility factor is absent.
• It contains ampicillin resistance
gene.
• It contains Lac promoter and
lac Z gene.
• The lac Z gene it contains 10 to
15 restricted sites for different
REN. It is called MCS ( multiple
cloning site.)

• multiple cloning site. (MCS): 10 to 15
restricted sites for different REN present in lac
Z gene of pUC 18 plasmid is called MCS.
Enzymes in genetic engineering:
• The two imp enzymes used as molecular
scissor and molecular stitchers are Restricted
endonuclease enzyme (REN) and DNA ligase.

Restricted endonuclease enzyme
• REN is the endonuclease enzyme that cuts
double stranded DNA molecule at specific
palindrome sequence. It is Used as a molecular
scissor in genetic engineering.
• REN are the defensive enzyme for bacteria. It
cuts and destroys bacteriophage DNA that infects
bacterial cell.
• Different types of REN are identified and isolated
for different palindromic sequence.

• Hamilton smith discovered and isolated
HIND II REN from Haemophilius influenzae in
1968. He received the Nobel Prize in
Physiology or Medicine in 1978.

palindromic sequence
• The region of DNA in which two strands are
identical when read in both the direction is
called palindromic sequence.
Ex: palindromic sequence for Eco-I is.

• palindromic sequence for HIND III is
5'-A |A G C T T-3'
3'-T T C G A| A-5‘
• In bacteria specific DNA palindromic sequence
are methylated periodically throughout the
genome. Hence REN is not effective against
bacterial genome.
• Foreign DNAs which are not methylated are
introduced into the cell are degraded by
sequence-specific restriction enzymes and
cleaved.

DNA ligase.
• The enzyme that joins the two sticky
ends of DNA is called DNA ligase. It is
used as molecular sitichers in genetic
engineering.
• DNA ligase was discovered by H G
Khorna.
• Dr. Hargobind Khorana was born on
9th January 1922 at Raipur, Punjab
(now in Pakistan).
• Died
November 9, 2011 (aged
89)Concord, Massachusetts, U.S.
• In 1968, He was awarded the Nobel
Prize in Physiology or Medicine for the
interpretation of the genetic code and
its function in protein synthesis

1922 - 2011

• Host cell: The cell to which desired new gene is
introduced is called host cell. Any living cell can be
used as host cell. Commonly E.coli bacterial cell is
used as host cell in genetic engineering. Because,
1. It is a simple prokaryotic cell.
2. It is a non-pathogenic bacteria.
3. It can be cultured easily in laboratory condition.
4. It has very short life span.
5. It contains self replicating plasmid.
6. The plasmid of E.coli can be easily handled as
vector.

Bioreactor
• It is an apparatus for culturing organisms like
algae, fungi, bacteria, or animal or plant cells
under controlled conditions.
• It is used in industrial processes to produce
pharmaceuticals, vaccines, or antibodies.
• It give the cells a homogeneous and controlled
environment by ensuring the same temperature,
pH, and oxygen levels.

Bioreactor Components
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Bioreactors consist of:
Vessel
Agitator
Sparger
inlets to maintain
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Temperature.
Dissolved Oxygen
pH
Pressure Gauge

• Ports for input and output of
material

• Bioreactor consists of
vessel which holds the
media and the cells. It can
be made of glass,
stainless steel, or a
durable plastic.
• An agitator or stirrer is
fixed inside to mix the
contents in the vessel.
Mixing of the contents is
to maintain a constant
nutrients and oxygen to
the culture.

• The sparger is an apparatus used
to introduce gasses into the
vessel. It aerate and supply
oxygen to the contents in the
vessel, as well as to the cells.
• Bioreactors has inlets to monitor
the culture in the vessel. Useful
inlets are foam control system
and pH control
• Cooling jacket with water
circulation maintains the
temperature.
• It contains additional ports to
introduce and remove materials
from vessels. The outlet is
present at bottom to collect
product.

Application of bioreactor.
• It is used to culture microbes like bacteria, fungi,
algae or plant cell or animal cell.
• It is used for the production of single cell protein.
• It is used for culturing genetically modified
microbes for production of antibiotics,
pharmaceutical compounds, vaccines etc.
• It is in the production of primary metabolites
from microbes.

Recombinant DNA technology:
• The technology of incorporation of desired gene
to the vector DNA and transferring it into host cell
is called r-DNA technology.
Steps involved in r-DNA technology:
1. Extraction of DNA or isolation of gene.
2. Selection of vector.
3. Gene splicing.
4. Transfer of r-DNA to the host cell.
5. Culturing of transformed host cell.

Extraction of DNA or isolation of gene:
• The cells of organism that
contains desired gene are
collected.
• The DNA of these cells is
extracted by using
refrigerated centrifuge
technology.
• The isolation of gene is done
by shoot gun method. In this
specific REN is used to cut
and isolate desired gene. The
isolated gene contains two
sticky ends.

• Complementary
DNA ( c-DNA)
• c-DNA is used
instead of isolating
desired gene. In
this m-RNA is
transcribed from
desired gene is
used as templet to
syntheses of DNA
using reverse
transcription.

• The single stranded DNA is later converted
into double stranded DNA.
• The DNA synthesized by reverse transcription
of m-RNA using reverse transcriptase enzyme
is called c-DNA.
• Artificial gene: The DNA synthesized with
reference to the number and sequence of
amino acids of protein chain in laboratory
condition is called artificial gene

• Multiplication of gene: The isolated desired
gene is multiplied into millions of copies using
polymerase chain reaction.

Selection of vector:
• Vector is a vehicle that carries
desired gene into host cell.
Depending on host cell vectors
like plasmids or phages are
selected.
Gene splicing:
• Incorporation of desired gene
into vector to develop r-DNA is
called gene splicing.
• The REN is used to cut the vector
at specific restricted site to insert
desired gene. Later it is ligated by
DNA ligase.
• The vector with desired gene is
called r-DNA.

Transfer of r-DNA into host cell:
• The bacterial cell ( host cell) and r-DNA are
made to suspend in cold (5-6 0C ) calcium
chloride solution. After some interval of time,
the temp of solution is suddenly raised to 42
0C and again cooled.
• The increase in temperature increases the
pore size of bacterial membrane. Through this
pore r-DNA enters the bacterial cell .

Culturing of transformed host cell:
• The transformed host cells are screened with
antibiotic to select r-DNA transformed cells.
These cells are isolated and cultured in
bioreactor.

Human insulin
• Insulin is a protein natured hormone that
maintains sugar metabolism. It converts the
excess of blood sugar (glucose) into glycogen
to maintain normal sugar level.
• This hormone is secreted by β-cells of islets of
Langerhans present in pancreas.
• The deficiency of insulin increases the blood
sugar level and causes diabetes mellitus.

• The diabetic patients are treated with
hypoglycemic oral drug or insulin injection.
• The oral drug stimulates the β–cells to secrete
insulin.
• In previous years insulin extracted from cows
and pigs are injected to control diabetic
condition. It causes allergy to most of the
patients.
• The r-DNA technology gives solution to
overcome this problem by producing human
insulin (humulin) using human insulin
producing gene.

Production of human insulin by r-DNA
technology.
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Tools required.
Proinsulin gene.
vector pUC 18.
REN HIND – III
DNA ligase.
Host cell- E.coli.
Bioreactor.

• m- RNA of proinsulin is used to produce
complimentary DNA by reverse transcription
process.
• The m-RNA of proinsulin is treated with
reverse transcriptase enzyme and deoxyribo
nucleotide to get c-DNA.
• Single stranded c-DNA hybridized to get
double stranded c-DNA.
• The c-DNA of proinsulin is incorporated with
pUC18 with in lac Z gene using REN HIND III
and DNA ligase.

• r-DNA and host cells E.coli are made to are
suspend in cold (5-6 0C ) calcium chloride
solution. After some interval of time, the temp of
solution is suddenly raised to 42 0C and again
cooled.
• The increase in temperature increases the pore
size of bacterial membrane. Through this pore rDNA enters the bacterial cell.
• The E.coli are screened to ampicillin to isolate
transformed cells.
• The transformed E.coli are cultured in bioreactor
to produce proinsulin.
• The transformed E.coli produces proinsulin along
with β–galactosidase.

• The fused proinsulin from β-galactosidase is
isolated by treating with cyanobromide
(CNBr).
• The proinsulin is inactive form and contains α,
β and c chain.
• It is treated with proteiolytic enzymes trypsin
and carboxy peptidase to remove c-chain
• The product obtained is functional insulin
having α and β - chains bounded by two disulphide bond.

Application of r-DNA technology.
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In production of human insulin to treat diabetes mellitus.
In production of growth hormone to treat dwarfism.
In production of blood clotting factor VIII to treat hemophilia.
In production of interferon's to treat viral disease and cancer.
In production of vitamins, enzymes, amino acids for
commercial use.
In production of alcohol.
In production of GMO plants as golden rice, BT plants, insect
resistance, viral resistance, plants.
In production of GMO microbes to clean environment
pollutant.
In production of GMO microbes to extract metals from low
grade ore.

DNA finger printing technology.
• The technology used for
identification of individual
at genetic level is called
DNA finger printing
technology.
• This technology was first
developed by alec
Jeffreys, Wilson and Thein
in 1985.

Born 9 January
1950 (age 62)
Oxford, United
Kingdom

• The principle is based on matching of VNTRs of
DNA collected at crime spot with suspect person
DNA.
• VNTRs: Variable number of tandem repeats. It is
also called as mini satellites.
• The identical and repeated sequence of
nucleotides present adjacent to each other in DNA
is called VNTRs.
• VNTRs are very specific to individual and differs
from person to person. It shows some similarities
between family members.
• VNTRs of identical twins are same. Hence it is not
possible to identify individuality in identical twins
by DNA finger printing technology.

Application of DNA finger printing
technology.
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It is used to identify criminals and rapist.
To solve parental dispute.
To solve immigrant problems.
To identify dead bodies of soldiers died in wars.
To identify dead bodies of person died at
accidents and bomb blast.
6. To identify racial groups.
7. To detect inheritable disorders.
8. To detect donor cell in case of transplantation.

Steps involved in DNA finger printing
technology.

• The DNA is isolated from the sample of blood
cells, hair root cells, semen or bone collected at
crime spot.
• The DNA of suspect also collected and isolated
separately.
• The isolated DNA is treated with REN to cut into
number of fragments.
• The DNA fragments are separated according to
their length on gel slab using gel electrophoresis.
• The DNA strand on gel slab is treated with
alkaline solution to split double strand in to single
strand.

• The single strand DNA is transferred to nylon
sheath using southern blotting technology.
• The single stranded DNA is hybridized with
radioactive probes of VNTRs . The excess of
probes are washed off.
• Nylon sheath is X-ray photographed to get
bands of VNTRs.
• The bands of X-ray sheath is the DNA finger
print.
• Comparing the DNA finger print of sample
collected at crime spot with suspect identifies
the individuality.

• Southern blotting: The technique of
transferring DNA from agar gel to
nylon sheath is called southern
blotting.
• Probe: Single stranded
polynucleotide fragment
complementary to specific sequence
of nucleotides of DNA is called
probe. It is mainly used in identify
VNTRs and desired gene

Gene therapy
• The technique of replacement of defective gene
by normal functional gene to treat genetic
disorder is called gene therapy.
Two ways of gene therapy:
1. Invivo approach.
2. Invitro approach.
• In invivo approach normal functional gene is
directly transferred to the target organ of patient.
• In invitro approach the defective cells are
cultured in lab condition. The normal gene is
transferred to this cultured cell.
The genetically modified cell or tissue is
transplanted to patient.

The disease cured by gene therapy;
1. SCID: Sevier combined immune deficiency
syndrome.
2. Cystic fibrosis.
3. Muscular dystrophy.
Types of gene therapy:
• Somatic gene therapy: It is the replacement of
defective gene by normal gene to somatic cells. It
is non heritable.
• Germ line gene therapy: It is the replacement of
defective gene by normal gene to germ cells. It is
to be done to avoid the inheritance of defective
gene to the next generation.

Methods of gene therapy
1. Viral method.
2. Non viral method.
• Viral method: in this method retrovirus are mainly
used as vector to transfer functional gene to the
target cell.
Steps involved in the viral method are,
• Selection of specific retrovirus that infect target cell.
• The virus used as vector is trimmed by removing
harmful pathogenic genes.
• The desired gene is incorporated into the vector.
• The vector is made to infect the target cells.

• Non viral method: Number of non viral methods
are used to transfer functional gene. Some of
them are,
1. Microinjection.
2. Electroporation.
3. Calcium phosphate mediated transfer.
• Micro injection: In this method functional gene is
directly transferred to target cell using micro
injection.

• Electroporation: In this method isolated cells
are subjected to low voltage electric shock. It
causes cell membrane to become permeable
for exogenous DNA.
• Calcium phosphate mediated transfer: In this
method functional gene is mixed with calcium
phosphate. This mixture is introduced near
target cells. The calcium phosphate disturbs
the cell membrane and makes permeable to
exogenous DNA

Embryo gene therapy through IVF-ET
( invitro fertilization and embryo transfer)
In this method gene therapy is given to the embryo through
IVF-ET. The steps involved are,
1. The gamete ovum and sperm are collected from defective
gene carrier patients.
2. The gametes are made to undergo fertilization invitro
under laboratory condition.
3. The zygote formed is incubated for three days to develop
in to 8cell stage.
4. The defective gene of the embryo cells are replaced by
normal functional gene.
5. The embryo is implanted back to the mother uterus for
further development.
6. The baby born is free from genetic disorders.

Monoclonal antibodies. (MABs).
• The specific antibody produced against
specific mono antigen artificially from
hybridoma cells is called monoclonal
antibodies.
• The hybridoma cells are developed by fusion
of B-lymphocytes and myeloma cells.

Steps involved in production of monoclonal
antibodies.
• The specific mono antigen to which antibodies
are required is injected to the mouse.
• The mono antigen stimulates the immune cells
to produce specific antibody.
• The B-lymphocytes that produces the specific
antibody are isolated from the spleen of
mouse.
• The isolated B-lymphocytes and myeloma cells
( tumor cells or cancer cells) are made to
suspend in polyethylene glycol (PEG) solution.
In this media two cells fuses and develops in to
hybridoma cells.

• The hybridoma has the capacity to undergo
uncontrolled mitotic cell division. These cells are
allowed to undergo multiplication.
• The hybridoma cells are screened for the ability of
monoclonal antibody production.
• The hybridoma cells that produces monoclonal
antibodies are cultured in hypoxanthin aminopterin
thymidine (HAT) for production of MABs.
• Some of the cells are frozen for future use.
Note:
1. PEG – Polyethylene glycol.
2. HAT- Hypoxanthin aminopterin thymidine .
3. ELISA - Enzyme linked immune sorbent assay.
4. RIA - Radio immune assay.

Application of monoclonal antibodies.
1. MABs are used for identification of cancer cells,
pathogens, enzymes, hormone assay etc.
2. It is used in ELISA and RIA to measure circulating level of
hormones and enzymes.
3. The specific MAB is used for identification of HIV by ELISA
test.
4. It is used to Identify pregnancy by assaying pregnancy
hormone HCG in urine.
5. It is used to identify A, B, AB and O blood groups.
6. It is used to identify sexually transmitted diseases.
7. It is used to treat cancer.
8. It is used as immune suppresser in organ transplantation.
9. Herceptin: Genetically engineered monoclonal antibody
used to treat breast cancer.

• U.S. govt. started Human genome project in 1986
coordinated by the Department of Energy and the
National Institutes of Health.
• GENOME – The whole hereditary information of an
organism that is encoded in the DNA is called
genome.
Aims or goal of the project:
• To identify the approximate 35,000 genes in the
human DNA.
• To determine the sequences of the 3 billion bases
that make up human DNA.
• To store this information in databases.
• To develop tools for data analysis.
• To address the ethical, legal, and social issues that
arise from genome research.

Achievement of HGP
• The project achieved to identify 35000 genes in
human beings.
• They sequenced about 3.2 billion base pairs in 23
pairs of chromosome
• Almost all (99.9%) nucleotide bases are exactly
the same in all people.
• The functions are unknown for over 50% of
discovered genes.
• Chromosome 1 has the most genes (2968), and
the Y chromosome has the fewest (231)

Application or Benefits of HGP
• It helps in understanding human genome
biology and human genetics.
• It helps to identify the gene associated with
genetic disorders.
• It helps in improving medicine and drugs.
• It helpful in giving gene therapy.
• It helps in studying human migration and
evolution.

Improvement of crop plants
• India planed green revolution in 1968 to over
come from the problem of scarcity of food and
starvation.
• It achieve its aim in 1978.
• Dr. swaminathan, Dr. w.K.Jain. Dr. Partha sarthi
and other agricultural scientist contributed
their work in green revolution.
• In this project crop plants are improved as
high yielding, disease resistance drought
resistance etc.

Indian organization for crop
production.
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IARI – Indian Agricultural research Institute.
ICAR – Indian council of Agricultural research.
CRRI – Central Rice Research Institute.
GKVK – Gandhi Krushi vignana Kendra.
DAU – Darwad Agricultural university

Plant breeding technique.
1. Introduction of crop plant.
2. Selection of crop plant.- Mass selection. pure
line selection, clonal selection
3. Hybridization.
4. Polyploidy breeding.
5. Mutation breeding.
6. Tissue culture and development of transgenic
plants.

• Hybridization: The cross made between two
plants differing in one or more desirable
characters.
• Intra specific hybridization: It is the cross made
between two plants of different breeds.
• Inter specific hybridization: It is the cross made
between two plants of different species but
belongs to same genus.
• Inter generic hybridization: It is the cross made
between two plants of different genus belongs to
same family.
• Usually inter specific and inter generic hybrids are
sterile. Polyploidy is induced to develop them
into fertile.

• Polyploidy breeding: The organisms having more
than one set of chromosomes are called
polyploidy. It is induced by spraying colchicine on
seeds or seedlings.
• Mutation breeding: Any change in chromosome
or chromosome number or sequence of DNA
leads to mutation.
• Dr. Swamynathan was the first person introduced
mutation breeding in India. Hence he was
regarded as father of radiation genetics in India.
• Mutation is induced by irradiating seedlings to xrays, ϒ-rays, α-rays, β-rays. Etc.
• It is also induced by exposing seedlings to
mustered gas.

Tissue culture and development of
transgenic plants.
• It is the technique of culturing cells into tissue, organ or
organism on cultural media under laboratory condition.
• Totipotency: The ability of a single cell develop into a
tissue or organ or individual is called totipotency.
• The totipotency of plant cells are more than animal cells.
• Explant: Any part of the plant body or tissue that is used
in tissue culture is called explant.
• Usually parenchyma tissue of stem or root is used as
explant.
• Callus: the undifferentiated and unorganized mass of
cells developed by explant during tissue culture is called
callus.

Requirements for tissue culture:
• Sterilization room: this room is used to
sterilize glass equipment's and explant. It is
also used to prepare media.
• Incubation room: It is germ free room. The
room is completely sterilized by using
luminous flow bench. In this room explant is
inoculated into culture media.
• Culturing room: In this room culturing tubes
are stored. The room is maintained by proper
temp, light and optimum humidity.

Steps in tissue culture.
• Sterilization: The sterilization of laboratory
equipment's is done by washing with potassium
dichromate solution. Further sterilization is done
by dry heat or autoclave.
• Preparation of media: The culture media is
prepared as formulated by scientist. The media
contains macro and micro nutrients. Essential
amino acids. Vitamins. Salt and some plant
hormones.
• Selection of explant: The explant is a small piece
of plant tissue. The parenchyma cells of stem,
root, apical bud meristem, or pollen grains are
used.

• Formation of callus: the explant selected is
sterilized and cut into number of pieces. Each
piece is inoculated into test-tube contain culture
media.
• Inoculated explant undergoes dedifferentiation
and develops in to mass of undifferentiated tissue
called callus.
• Inducing organogenesis: the organogenesis is
induced by applying different ratio of plant
hormones - Auxin and cytokinin.
• After organogenesis the seedlings having small
roots and shoot is transferred to plastic bags
containing fertile soil.
• These are grown in to small plants under green
chamber. Later plants are transferred into fields.

Application of tissue culture.
• Micropropogation –production of millions of
plants by tissue culture. It is applied to
increase number of crop plants, medicinal
plants, forest plants, endangered plants.
• Production of haploid plants: anther or pollen
grain culture results in development of
haploid plant. The chromosomal dabbling is
done to get diploid or polyploidy.
• Production of viral free plants: The apical bud
culture is done to develop viral free plant.

• Production of sec metabolic compounds: the callus
culture is transferred to bio reactor for production
of pharmaceutical compounds, alkaloids, colouring
agent etc.
• Production of transgenic plants: the desired gene is
introduced into cells of callus to develop into
transgenic plants.

Transgenic plants.
• The genetically modified plants developed by transferring
desired gene are called transgenic plants.
• Some transgenic plants are,
1. nif plants involve in nitrogen fixation.
2. BT plants having pest resistance.
3. Golden rice plant that fulfills vitamin A deficiency.
Importance of transgenic plants.
• Transgenic plants are developed,
1. To improve crop plants for high yield.
2. To produce disease resistance plants.
3. To produce pest resistance.
4. To produce drought resistance.
5. To produce secondary metabolite’s.

Golden rice:
• It is genetically modified
rice plant which is rich in
beta carotene a precursor
of vitamin A in its
endosperm.
• The golden rice was first
developed by Ingo Potrykus
(1999). The IR-64 rice is
selected to develop golden
rice.

• The important genes transferred
to IR-64 are,
• PSY – Phytoene synthatase.
• LYC – Lycopane cyclase. From
Daffodil plant.
• Ctrl-I gene – to synthesis
enzymes of the biosynthetic
pathway of b-carotene from
Erwinia uredovora.

Steps in development of golden rice.
• The two specific genes PSY and LYC that involves in
production of b-carotene are isolated from daffodil plant.
• ctrl-I gene that synthesizes necessary enzyme for
production of provitamin A is isolated from the bacteria
Erwinia uredovera.
• These three genes are incorporated into Ti-plasmid to
develop r-DNA.
• This r- DNA is first transferred to bacteria Agrobacterium
tumefaciens.
• These bacteria are cultured to get number of cloned genes.
• The transformed agrobacterium is made to infect IR- 64 rice
embryo.
• the infected embryos are screened for transformed genes
and cultured.
• The seedlings produced from these cultured embryos are
called Golden rice.

• The seeds of golden rice are golden yellow in
colour. The density of colour depends upon
richness of b-carotene.

Improvement of animals.
• Animal husbandry: It is the science of raring
breeding and caring of domestic animals ( live
stock).
• Number of methods are applied to improve
animals using the knowledge of genetics and
reproductive physiology.
• The cattle breeds are mainly improved by cross
breeding.
• Cross breeding: mating of two parental animals of
different breeds to develop a hybrid is called
cross breeding.

In cattle's it is applied to develop a hybrid in such a way
that,
1. To increase the capacity of milk production.
2. To increase lactation period up to 10 months.
3. To increase reproductive capacity.
4. To develop resistance to disease.
5. To make them to adopt tropical and sub-tropical
climates.
• Artificial insemination: it is injecting the semen of
desired bull into female reproductive tract
mechanically.
• Cryopreservation: It is preservation of semen in
liquid nitrogen at -70 C to -196 C. The frozen semen
can be stored up to 20 years.

Advantage of artificial insemination.
• The semen collected from single ejaculation of
bull can be inseminated to five hundred cows.
• The semen can transmitted easily than
transporting bull.
• The frozen semen can be stored upto 20 years.
Hence there is no need of maintaining large
bull yard.

MOET - multiple ovulation and embryo transfer
(OR) SOET - super ovulation and embryo transfer.
• Super ovulation is the
technique in which female
cow is forced to release
large number of ovum by
injecting Follicle
stimulating hormone.
• Embryo transfer is a
technique of transferring
eight cell stage embryo to
surrogate mother for
further development.

• Steps involved in SOET or
MOET.
• Estrus synchronization: The
donor and surrogate mother
cows are artificially made to
have same reproductive stage. It
is done by injecting gonado
tropic hormone.
• Super ovulation: The follicle
stimulating hormone is injected
to donor cow to release large
number of eggs.

• Artificial insemination: The
super Ovulated donor cow is
artificially inseminated by
desired bull semen.
• Embryo recovery: After
fertilization eight cell
embryos are recovered for
further development by
surgical or non-surgical
method.

• Embryo transfer: The collected embryos are
transferred to surrogate mother cow uterus
for further development.
• By this technique desired breed yard can be
increased in short period.

• IVF- ET : Invitro fertilization and embryo
transfer:
• In this technique superovulated ovum are
collected and fertilized in laboratory
condition.
• The fertilized ovum are incubated to develop
into eight cell stage.
• These embryos are transferred to surrogate
mother for further development.

Stem cells:
• The undifferentiated cells that have the ability
to undergo mitosis and differentiation in to
tissue are called stem cells.
• The two types of stem cells are
1. embryonic stem cells
2. Adult stem cells.
• The inner masses of cells of blastula are
embryonic stem cells.
• The bone marrow cells, placental cells are the
adult stem cells.

Application of stem cell culture:
• Cultured stem cells are used to differentiate into
different types of cells like liver cells, nerve cells,
muscle cells and blood cells etc.
• The differentiated tissue cells are used to treat
nervous disorders like Parkinson’s disease,
Alzheimer’s disease spinal cord injury, etc.
muscular destropy, cardiovascular disorders are
also treated.
• Cultured stem cells are used in genetic
modification.
• Stem cells are used to repair damage defective
tissue.

Hazards of biotechnology.
• Genetic engineering may develop new pathogen
• Genetically modified microbes may use as
biological weapons.
• Many transgenic food causes allergy in few
people.
• BT toxin transgenic plants kills and decreases the
population of different useful insects.
• Genetically modified crop plants for resistance to
weedicide can cross naturally with weeds. It
becomes difficult to control weeds.
• Wide spread of transgenic plants depletes the
agriculture biodiversity.
• Animal genes transferred to crop plants arises
ethical and religious problems.

Safeguard of genetic engineering.
• Strict laboratory procedure should follow in
biotechnology labs.
• Genetically engineered microbes are crippled in
such a way that they cannot survive outside the
laboratory condition.
• The permission from r-DNA advisor committee is
taken to undergo genetic work.
• The permission from genetic engineering
committee is taken to release genetically modified
organism.
• Human cloning and transgenic human experiments
should be banned.

Biotechnology MohanBio

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