ASWINI SIR’S

BIOTECHNOLOGY AND ITS APPLICATIONS

➢ Biotechnology refers to an integrated applied branch of science, which is useful to humans in
producing various products by using microbes, plants animals and their metabolic machinery.
➢ This is achieved by recombinant DNA technology, in which valuable and useful manipulation
of genes in various living species is done to obtain desirable products at a large scale.

APPLICATIONS IN AGRICULTURE AND FORESTRY

➢ Plants genetic transformation has become an important biotechnology tool for the
improvement of crops. A solid foundation for the rapid development and implementation of
biotechnology in agriculture has been laid by plant culture.
Plant Cell and Tissue Culture:
➢ Plant cell and tissue culture has been incorporated with plant genetic engineering by giving
rise to a new branch known as plant biotechnology.
➢ Plant genetic engineering involves the manipulation of the plant genome by transferring a
beneficial gene from one plant cell to another and then growing the transformed plant cell in
an artificially enriched media.
➢ The totipotent cell divides. and re-divides and forms a mass of undifferentiated cells that
constitute a callus.
➢ On appropriate hormonal stimulation, rooting and shooting takes place and the callus turns
into a plantlet.
Drought Resistant Plants:
➢ Plant tissue culture technique is helpful in generating drought resistant plants by introducing
those genes whose products enable the plants to retain water and withstand prolonged
drought conditions.
Fungi and Bacteria Resistant Plants:
➢ The genes encoding certain enzymes and proteins which give resistant abilities to a plant can
be introduced into isolated plant cells in a culture media to generate fungi and bacteria
resistant plants.
Virus Resistant Plants:
➢ Transgenic plants have been generated, which express the coat protein genes of infectious
plant viruses. The coat protein, thus expressed, turns on the plant version of the immune
system. Thus, the expressed coat proteins function as plant vaccines.
Herbicide Resistant Plants:
➢ Herbicide resistant transgenic plants have been generated by transferring bacterial herbicide
resistant genes into plant cells grown in culture.
➢ Glyphosate is the most widely used broad-spectrum herbicide world over.
➢ A glyphosate resistant gene from Petunia plant is transferred into isolated plant cells in culture
and glyphosate resistant plants generated.
➢ Weeds such as Striga which decrease crop yield and quantity can be removed with the help of
herbicide (weed killer), e.g., Roundup Ready transgenic plant has been produced and
commercialised as it is tolerant to the herbicide Roundup (Trade name).
Frost (Ice) Protected Plants:
➢ Crops grown at sub-zero temperature are subjected to frost damage due to formation of ice
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crystals in their tissues.

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➢ Soil bacterium Pseudomonas syringae contains a gene that promotes ice nucleation. Using
gene technology, this gene is deleted and a mutant P. syringae is engineered known as INA or
ice-minus strain which prevents frost (ice) formation when sprayed on crops.
Bioplastic:
➢ An alternative to natural plastic called bioplastic, has been developed using biotechnology.
➢ Certain group of microorganisms synthesize biopolymers similar to natural plastic.
➢ The gene encoding the enzyme for biopolymer synthesis is isolated and transferred to corn
plant cells in culture and transgenic corn plants generated, which express the transgene and
synthesize the biodegradable polymer which is used as bioplastic. It does not cause
environmental pollution.
Novel Transgenic Plants:
➢ In Petunia, a gene that codes enzymes for flower colour was introduced in protoplasts and a
trans genic plant with pink flowers was generated.
➢ Florigene, a biotech company in 1996 released the first ever genetically manipulated flower
into the market
➢ Another example of the novel plant is the luminescent tobacco plant which is generated
through gene manipulation.
➢ The luciferase gene of bacteria or firefly expresses luciferase enzyme. It acts on the substrate
luciferin and turns it into a product, which glows in dark. This phenomenon is known as
bioluminescence.
➢ The tobacco plant cells in culture are transformed by a recombinant Ti plasmid containing a
luciferase gene. Plants, generated, glow in dark in the presence of the substrate luciferin.
Protein Producing Plants:
➢ Transgenic plants also serve as bioreactors for the synthesis of many therapeutic mammalian
proteins such as enkephalin (a neuro-peptide) and human serum albumin.
Golden Rice:
➢ It is a genetically modified rice that contain increased level of β-carotene (a precursor of
vitamin-A) which is produced by introducing genes encoding the enzymes of β-carotene
biosynthetic pathway into rice plant cells in culture.
➢ It is light yellow in colour and helps to prevent night-blindness in the world.
Delayed Fruit Ripening (Flavr Savr Tomato):
➢ Usually, tomato is a food item which is consumed everywhere in the world. It is a seasonal
vegetable and persists for a short duration due to its fast ripening and rotting.
➢ The ripening of tomato occurs due to the synthesis of polygalacturonase enzyme which
degrades the pectin of tomato.
➢ By the application of biotechnology, the polygalacturonase enzyme was blocked so that the
ripening and rotting of tomato is delayed.
➢ These genetically engineered tomatoes are known as Flavr-Savr tomato.
Nitrogen-Fixation:
➢ The atmospheric nitrogen is trapped by soil microorganisms and converted into soluble nitrate
by a process, called biological nitrogen fixation. This process is catalysed by an enzyme
complex, nitrogenase, encoded by nitrogen fixing (nif) genes.
➢ The nif gene can be transferred from the genome of Rhizobium into the chromosomes of non-
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leguminous plants, so that they can utilize atmospheric nitrogen.

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GENETICALLY MODIFIED ORGANISMS (GMO)

➢ GMOs are the plants, bacteria, fungi & animals whose genes are altered by manipulation.
These are also called transgenic organisms as they contain and express one or more foreign
genes called transgenes.
Advantages of Genetic Modification in Plants:
➢ It makes crops more tolerant to abiotic stresses (cold, drought, salt, heat etc).
➢ Pest-resistant crops reduce the use of chemical pesticides.
➢ It helps to reduce post-harvest losses.
➢ It increases efficiency of mineral usage by plants, preventing early exhaustion of fertility of
soil.
➢ It enhances nutritional value of food. E.g., Vitamin ‘A’ enriched rice (golden rice).
➢ Genetic modifications are also used to create tailor-made plants to supply alternative resources
to industries, in the form of starches, fuels and pharmaceuticals.

Pest Resistant Plants

➢ Pest Resistant Plants act as bio-pesticide. These reduce the need for insecticides.
➢ E.g., Bt cotton, Bt corn, rice, tomato, potato, soya bean etc.

Bt Cotton:
➢ Some strains of bacterium Bacillus thuringiensis have proteins that kill insects like coleopterans
(beetles), lepidopterans (tobacco budworm, army worm) & dipterans (flies, mosquitoes).
➢ B. thuringiensis forms a toxic insecticidal protein (Bt toxin) crystal during a particular phase of
their growth. These crystals do not kill the Bacillus as it exists as inactive protoxins.
➢ When an insect ingests the inactive toxin, it is converted into active toxin due to the alkaline
pH of the gut which solubilise the crystals. The active toxin binds to the surface of mid gut
epithelial cells and creates pores. It causes cell swelling, lysis and finally death of the insect.
➢ These Bt toxin genes were isolated from B. thuringiensis and incorporated into crop plants such
as cotton.
➢ Most Bt toxins are insect-group specific. This toxin is coded by a gene called cry.
➢ E.g.,
✓ The proteins encoded by the genes cry IAc and cry IIAb control the cotton bollworms
✓ cry IAb control corn borer.
✓ cry IIIAb controls Colorado potato beetle
✓ cry IIIBb controls corn rot worm.

Nematode Resistance in Tobacco Plants:

➢ A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great
reduction in yield.
➢ RNA interference (RNAi) strategy is used to prevent this infestation.
➢ RNAi is a method of cellular defence in all eukaryotic organisms. It prevents translation of a
specific mRNA (silencing) due to a complementary dsRNA molecule.
➢ The source of this complementary RNA (dsRNA) is from an infection by viruses having RNA
genome or mobile genetic elements (transposons) that replicate via an RNA intermediate.
➢ Using Agrobacterium vectors, nematode-specific genes (DNA) were introduced into the host
plant. It produced both sense & anti-sense RNA in host cells.
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➢ These two RNAs are complementary to each other and formed a double stranded (dsRNA)
that initiated RNAi and thus, silenced the specific mRNA of nematode.
➢ Thus, the parasite cannot survive in a transgenic host expressing specific interfering RNA.

APPLICATIONS IN MEDICINE
➢ The recombinant DNA technology helps for the mass production of safe and more effective
therapeutic drugs.
➢ The recombinant therapeutics does not induce unwanted immunological responses as is
common in case of similar products isolated from non-human sources.
➢ At present, about 30 recombinant therapeutics have been approved for human-use. In India,
12 of these are presently being marketed.

Genetically Engineered Insulin:

➢ Management of adult-onset diabetes is possible by taking insulin at regular time intervals.
➢ Previously, insulin was extracted from the pancreas of animals (cattle & pigs) which caused
allergy or other types of reactions due to the foreign
protein.
➢ Now, it is possible to produce human insulin using
bacteria.
➢ Insulin consists of two short polypeptide chains (chain
A & chain B) that are linked together by disulphide
bridges.
➢ In mammals, insulin is synthesized as a pro-hormone.
The pro-hormone needs processing before it becomes
a fully mature and functional hormone.
➢ The pro-hormone contains an extra stretch called the C peptide. This is removed during
maturation into insulin.
➢ Therefore, the main challenge for production of insulin using rDNA technique was getting
insulin into its mature form.
➢ In 1983, Eli Lilly, an American company prepared two DNA sequences corresponding to A &
B chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains.
These chains (A & B) were produced separately, extracted and combined by creating
disulphide bonds to form human insulin called Humulin.
➢ Now a days, humulin is used for treatment of diabetes mellitus.

Recombinant Vaccine:
➢ The vaccines produced through genetic engineering methods are called recombinant vaccines
or second-generation vaccines.
➢ They have gene inserts for the surface proteins of a pathogen that bring out immunity but do
not result in infection
➢ These plasmids are inserted in bacteria or yeast cells that express the viral proteins which are
then injected into human host as vaccine.
➢ E.g., Hepatitis-B vaccine, Polio vaccine
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Stem Cell:
➢ These are undifferentiated biological cells that can differentiate into specialized cells and can
divide through mitosis to produce more stem cells.
➢ These are mainly of 2 types. i.e., embryonic stem cell (isolated from inner cell mass of
blastocyst) and adult stem cell (exist throughout the body after embryonic development
including brain).
➢ The potential applications of stem cell include organ and tissue regeneration, bone marrow
transplantation, brain disease treatment, blood disease treatment etc.

Gene Therapy
➢ It is a method to correct a gene defect (hereditary disease) diagnosed in a child or embryo.
➢ It can be achieved by the following methods.
i. By replacement of a defective gene with a normal gene.
ii. By correcting a defective gene through gene targeting.
iii. By gene augmentation, i.e., normal foreign gene sequences are introduced for defective
gene.
➢ First clinical gene therapy was attempted in 1990 on a 4-year-old girl with adenosine
deaminase (ADA) deficiency by M Blease and WF Andresco of National Institute of Health.
➢ The disorder is caused due to the deletion of the gene for adenosine deaminase (the enzyme
crucial for the immune system to function).
➢ This can be cured by bone marrow transplantation or by enzyme replacement therapy
(injection of functional ADA). But these approaches are not completely curative.
➢ This is why gene therapy was introduced.
➢ Steps in enzyme replacement gene therapy:
✓ Lymphocytes from the patient’s blood are grown in a culture outside the body.
✓ A functional adenosine deaminase cDNA (using a retroviral vector) is introduced into
these lymphocytes.
✓ Then, they are returned to the patient. This should be periodically repeated as these cells
are not immortal. However, if the ADA gene (from marrow cells) is introduced into cells
at early embryonic stages, it could be a permanent cure.
➢ Some other diseases that can be treated by gene therapy are haemophilia, cystic fibrosis etc.

Molecular Diagnosis:
➢ Recombinant DNA technology, PCR (Polymerase Chain Reaction) and Enzyme Linked
Immuno-Sorbent Assay (ELISA) are some techniques for early diagnosis.
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Polymerase Chain Reaction (PCR):

➢ It helps in early detection of a disease or pathogen.
➢ The presence of a pathogen is normally suspected only when the pathogen has produced a
symptom. By this time the concentration of pathogen is already very high in the body.
➢ However, very low concentration of a bacteria or virus can be detected by PCR by
amplification of their nucleic acid.
➢ PCR is used to detect HIV in suspected AIDS patients. It is also used to detect mutations in
genes in suspected cancer patients. It is a powerful technique to identify many other genetic
disorders.
➢ Steps of PCR:
✓ A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to
hybridise to its complementary DNA in a clone of cells
✓ The cells are then detected by autoradiography.
✓ The clone having the mutated gene is not observed on the photographic film, because the
probe will not have complementarity with the mutated gene.

Enzyme Linked Immuno-Sorbent Assay (ELISA):

➢ This technique is commonly used for the diagnosis of AIDS, hepatitis, TB, rubella virus
infection, thyroid disorders, pregnancy test through presence of hCG in urine or blood and
other sextually transmitted diseases.
➢ It is a very sensitive immunological and serological test which based on the principle of
antigen-antibody interaction.
➢ This technique can detect extremely small amount of a protein, antibody or antigen with the
help of enzymes such as peroxidase and alkaline phosphatase.
➢ There are special substrates for enzymes which produce coloured products like 5-
aminosalicylic acid, orthophenylene diamine etc.
TRANSGENIC ANIMALS

➢ The term ‘transgenic’ was first given by Gorden and Ruddle in 1983.
➢ These are the animals whose genome has been altered by introduction of an extra (foreign)
gene by manipulation.
➢ The process by which these animals are formed is called transgenesis.
➢ E.g., Transgenic rats, rabbits, pigs, sheep, cows and fish.
➢ Over 95% of all existing transgenic animals are mice (supermouse).
➢ In 1996, cloning was revolutionized when Ian Wilmut and his colleagues at the Roslin-
Institute in Edinburgh, Scotland, successfully cloned a sheep named Dolly. Dolly was the first
cloned mammal.

Benefits of Transgenic Animals

➢ To study normal physiology & development:
✓ Transgenic animals are used to study how genes are regulated, and how they affect the
normal body functions and its development.
✓ E.g., Study of complex factors involved in growth such as insulin-like growth factor.
✓ Genes (from other species) that alter the formation of this factor are introduced and the
biological effects are studied.
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✓ This gives information about the biological role of the factor in the body.
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➢ To Study the contribution of genes in the development of a disease:

✓ Transgenic models help for investigation of new treatments for human diseases.
✓ E.g., transgenic models for many human diseases such as cancer, cystic fibrosis,
rheumatoid arthritis and Alzheimer’s.
➢ Biological products:
✓ Some medicines contain biological products, but they are often expensive.
✓ Transgenic animals are used to produce useful biological products by introducing genes
which codes for a particular product.
✓ E.g., Human protein (α-1-antitrypsin) used to treat emphysema, similar products for
treatment of phenylketonuria (PKU) and cystic fibrosis etc.
✓ In 1997, Rosie (first transgenic cow) produced human protein-enriched milk (2.4 gm per
litre).
✓ It contains the human α-lactalbumin and is nutritionally more balanced product for
human babies than natural cow-milk.
➢ Vaccine safety testing:
✓ Transgenic mice are used to test the safety of vaccines before they are used on humans.
E.g., Polio vaccine.
✓ If it is found to be reliable and they can replace the use of monkeys to test the safety of
batches of the vaccine.
➢ Chemical safety testing (toxicity testing):
✓ Transgenic animals are made that carry genes, which make them more sensitive to toxic
substances than non-transgenic animals.
✓ They are exposed to the toxic substances and the effects studied. It gives immediate results.

Note: Bio-safety- Biosafety is defined as, “The discipline addressing the safe handling and
containment of infectious microorganisms and hazardous biological materials”. In India,
Department of biotechnology is the nodal centre for Indian biosafety network.

ETHICAL ISSUES
1. Problem of unpredictable results:
➢ Genetic modification may cause unpredictable results when such organisms are introduced
into the ecosystem.
➢ Therefore, there is a need for some ethical standards to evaluate our actions. These set of
standards that are used to regulate activities in relation to biological world are called bioethics.
➢ In order to control these issues, Indian Government has set up organizations like GEAC
(Genetic Engineering Approval Committee), which make decisions about the validity of GM
research and the safety of GM-organisms for public services.

2. Problems of Patent:
Patent:
➢ It is a set of exclusive rights granted by a government to an inventor or their assignee for a
limited period of time to prevent others from commercial use of their invention.
➢ Patents are supposed to satisfy three criteria of: Novelty, Non-obviousness, and Utility.
➢ Novelty implies that the innovation must be new. It cannot be part of ‘prior art’ or existing
knowledge.
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➢ Non-obviousness implies that it may not be documented but is otherwise well known.

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➢ The discorded fact or product should be of a particular use for the human beings.
➢ When the patent is granted for biological entities and for products derived from them, they are
called bio-patents.
➢ Primarily, USA, Japan and members of European Union are awarding bio-patents.

Controversies in India regarding patent and biopiracy:

➢ Certain companies have got patents for products and technologies that make use of the genetic
materials, plants etc. that have been identified, developed and used by farmers and indigenous
people of a specific country.
➢ E.g., Basmati rice, herbal medicines like turmeric, neem etc.
➢ Diamond vs Chakraborty Case: Dr. Anand Mohan Chakraborty, an Indian born American
scientist, developed a bacterium species of Pseudomonas (also known as superbug) that could
eat the oil and consequently clear oil spill. He applied for a patent for this bacterium, but was
denied by patenting authorities on the plea that patent could not be granted because the
bacterium was a living organism. Then, he moved to supreme court. After several hearings, in
1980 court ruled in favour of Chakraborty.
➢ Basmati Rice Patent Case: Basmati rice has unique aroma & flavour, whose 27 varieties are
cultivated in India. In 1997, an American company (Rice Tec) got patent rights on Basmati rice
through the US Patent and Trademark Office (USPTO). This allowed the company to sell a
‘new’ variety of Basmati in US and Abroad. This new variety was actually derived from Indian
farmer’s varieties. Indian Basmati was crossed with semi-dwarf varieties and claimed as an
invention or novelty by the American company. However, due to people’s movement against
Rice Tec, USPTO partially rejected the patent.
➢ Turmeric Patent Case: In May 1995, a US patent on turmeric was granted to two US based
Indians (University of Mississippi Medical Centre), especially for the use of turmeric powder
in wound healing. Two years later, a complaint was filed by Dr. R A Mashelkar of Council of
Scientific and Industrial Research (CSIR) challenging the novelty of invention. The validity of
the patent was examined and finally, in 1997, the patent was cancelled.
➢ Neem Patent Case: A patent was granted by European Patent Office (EPO) to the multinational
agribusiness company, W.R. Grace of New York and United States Department of Agriculture,
Washington DC for ‘fungicidal uses of neem oil’. It was stated that neem oil controlled fungal
growth on plants. Dr. Vandana Shiva of Research Foundation for Science and Technology &
Natural Resource Policy, New Delhi and others challenged to the grant of this patent. The case
was admitted and after several round of hearing, the patent granted to W. R. Grace was
withdrawn in 2005.

3. Biopiracy:
➢ It is the use of bio-resources by multinational companies and other organizations without
proper authorization from the countries and people concerned (also without compensatory
payment).
➢ Most of the industrialized nations are financially rich but poor in biodiversity and traditional
knowledge.
➢ The developing and the underdeveloped world have rich biodiversity and traditional
knowledge related to bio-resources. Hence, these countries have to develop laws to prevent
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unauthorized exploitation of bio-resources and traditional knowledge.

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➢ Another cause of biopiracy is bioprospecting which means thorough survey of a source

material to expand the knowledge and applications in biotechnology. During the course of
bioprospecting, scientists may transfer any biological resource which they may consider as
novel.
➢ Ex. Neem patent case, Turmeric patent case etc.
➢ Indian Parliament has cleared the second amendment of the Indian Patents Bill that takes such
issues into consideration, including patent terms, emergency provisions, research and
development initiative.
Bio-war:
➢ The war in which bio-weapons in the form of bacteria, viruses or any other micro-organisms
are used is called bio-war.
➢ Bio-weapons are low cost and cause more casualties.
➢ Bio-terrorists often used these weapons to terrorize the innocent people.
➢ The disease-causing microorganisms can easily spread to long distance.
➢ During 1st world war Bacillus anthracis was used by the by Germany.
Transgenic mouse/ Super mouse: The first transgenic mouse was developed by R.L. Brinster and
R. Palmiter in 1982. They isolated growth hormone gene from a rat and transferred into a fertilized
mouse by microinjection in vitro. Then this fertilized egg was implanted in the uterus of
pseudopregnant mouse. The resulting progenies were called supermouse because pf their large
size and abnormal growth.

Pharming: It is the commercial use of transgenic animals as a source of important pharmaceutical

products. Ex. the supermouse was genetically altered to produce tissue plasminogen activator
(TPA), an agent that dissolves the blood clot. Several other pharmaceutical products like urokinase,
α-1 antitrypsin, insulin, growth hormone, blood coagulation proteins (factor VIII ad IX),
fibrinogen, lactoferrin (an infant nutrition formula) have been successfully harvested.

Animal cloning and making of Dolly: Ian Wilmut of Roslin Institute of Scotland came up with a
cloned sheep, named Dolly in February, 1997. He used nuclear transplantation technique to create
Dolly, a clone of sheep. This was the first ever clone of an animal. Following this, two other sheep,
named, Polly and Molly were also created in the same manner.

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Prepared By- Dr. Aswini Nayak, M.Sc., M.Phil., Ph.D Cont.-9439887779