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BTNY-MM: XII 10. BIOTECHNOLOGY & ITS APPLICATIONS
Biotechnology has a wide range application such as B. Bt-cotton
- Biopharmaceuticals, therapeutics and diagnostics Some insect larva attacks crops such as cotton.
- Genetically modified crops for agriculture and processing food
- For bioremediation (cleaning toxic chemicals present in soil Order Insects
by transgenic plants or microbes) Lepidopterans Tobacco budworm, armyworm
- Waste treatment, energy production etc. Coleopterans Beetles
Dipterans Flies, mosquitoes
APPLICATION IN AGRICULTURE It destroys the leaves or cotton boll which results in great
Advantages of Genetically Modified crops: reduction in yield.
Increased stress tolerance (against cold, drought, salt, heat etc).
Pest-resistant crops reduce the use of chemical pesticides.
Reduced post harvest loss.
Increased efficiency of mineral usage by plants.
Enhances nutritional value of food. E.g. Vit.‘A’ enriched
Golden rice. Traditionally we use insecticides to prevent insect attack.
Recently we modified the cotton plant using Bt toxin gene (from
A. Tissue Culture Bacillus thuringiensis) which itself produce insecticides.
Plant tissue culture is the technique of growing plant in an
artificial culture medium under sterile conditions. How B. thuringiensis resist insects?
It is based on the principle of cellular totipotency i.e., a plant Step 1. B. thuringiensis produces insecticidal protein crystals /
cell can generate a whole plant. Bt-toxin. (It does not kill the Bacillus itself as it exists as
inactive protoxins).
Step 2. Insect ingest the protoxin
Step 3. In the gut of insect, due to the alkaline pH, crystal protoxin
solubilise to form active toxin.
Step 4. The toxin binds to the surface of midgut epithelial cells
Procedure:- and creates pores.
(Step-1) Take an explant (any part of parent plant). Step 5. It leads to the death of the insect.
If it is a virus infected plant, meristems are used as explant
to obtain healthy plants from it. It is because meristems will
be free of virus because virus cannot reach at there due to
absence of vascular tissues
(Step-2) Grow the explant in a nutrient media containing sucrose, salts,
certain vitamins, amino acids and growth regulators (auxins,
cytokinins etc).
(Step-3) Thousands of plants produced in short duration (micropropagation).
These plants are genetically similar to parental plant so called How to develop insect-resistance in cotton?
somaclones. Step 1. Bt-toxin genes were isolated from B. thuringiensis.
Toxins producing cry genes are insect specific-
SOMATIC HYBRIDIZATION
Somatic hybridization is the fusion of protoplasts of 2 somatic cry gene Insect controlled
cells from different varieties of plants (with desirable characters) cryIAb Corn borer
to develop hybrid protoplasts. cryIAc
Cotton bollworms
Procedure:- cryIIAb
Step 1. Isolate a single cells from tomato and potato
Step 2. Incorporated into cotton.
Step 2. Digest their cell walls and isolate protoplasts
Step 3. Bt toxins produced by cotton plant, if attacked by insect, kill
Step 3. Fuse the protoplasts to obtain hybrid protoplasts, in tissue
them.
culture media.
Step 4. They regenerate cell wall and grown to form a new plant,
pomato (Somatic hybrid).
But Pomato has no desired characters for its commercial utilization.
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In 1983, Eli Lilly an American company prepared human insulin.
C. Nematode resistance in tobacco plants:
A nematode Meloidegyne incognitia infects the roots of tobacco
plants and causes a great reduction in yield.
The process of RNA interference (RNAi) is used to prevent this
infestation.
What is RNAi?
RNAi prevents / interfere the translation of a specific mRNA
(silencing) from the gene by binding of a complementary dsRNA
molecule.
How to RNAi works in tobacco?
(Step-1) Prepared 2 DNA sequences corresponding to A & B chains
of human insulin
(Step-2) Introduced them in plasmids of E. coli separately.
(Step-3) E. coli produced insulin chains A & B and extracted from it.
(Step-4) Chains are combined by creating disulfide bonds to form
human insulin.
B. Gene Therapy:
o Gene therapy is a method to correct a gene defect diagnosed in
a child/embryo.
By inserting normal gene, the defective genes are replaced or
non-functional genes are compensated.
o First clinical gene therapy was given in 1990 to Ashanti
DeSilva of U.S, a 4-year old girl with adenosine deaminase
(ADA) deficiency.
It is due to the deletion of the gene for adenosine deaminase
(the enzyme crucial for the immune system to function).
(Step-1) Isolatenematode-specific gene from RNA viruses or mobile
genetic elements (transposons).
(Step-2) Introduce nematode-specific genes into the tobacco plant
using Agrobacterium.
(Step-3) It produces both sense & anti-sense RNA in host cells.
These 2 RNA’s being complementary to each other formed a
dsRNA which silence the specific mRNA of nematode
(RNAi) which is responsible for the synthesis of protein
which is essential for the growth of the nematode.
(Step-4) The parasite cannot survive in a transgenic host and die. This can be cured by –
1. Bone marrow transplantation
APPLICATION IN MEDICINE 2. Enzyme replacement therapy (injection of functional ADA).
Advantages of rDNA technology in medicine:- But a permanent cure can be achieved through gene therapy.
a. Enable the mass production of safe and effective drugs.
b. Since the recombinant therapeutics are identical to human proteins,
they do not induce unwanted immunological responses
A. Humulin (Genetically Engineered Human Insulin)
o In early days, insulin used for diabetes was extracted from
pancreas of slaughtered cattle and pigs. It caused allergy to some
patients.
But now we could make human insulin using bacteria.
Structure of Insulin-
Insulin consists of 2 short polypeptide chains that are linked
together by disulphide bridges.
(Step-1) Lymphocytes are collected from the patient
(Step-2) It is grown in a culture.
(Step-3) Introduce functional ADA cDNA into these lymphocytes
using a retroviral vector.
Insulin is synthesized as a pro-hormone containing an extra stretch (Step-4) Return the lymphocytes to the patient’s body.
called C-chain.
This should be periodically repeated as these cells are mortal.
The pro-hormone, after removal of C-chain (processing), becomes
a fully mature hormone. For permanent cure, the ADA gene (from marrow cells) is
introduced into cells at early embryonic stages.
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C. Molecular Diagnosis:
Molecular diagnosis is the techniques for early detection of
diseases even before detecting from urine or serum analysis. It
aids in effective treatment of a disease.
rDNA technology, PCR & ELISA are some of these techniques.
1. PCR (Polymerase Chain Reaction)
Very low concentration of a pathogen can be detected by
amplification of pathogen’s nucleic acid by PCR.
Applications of PCR
- It is a technique to identify many genetic disorders.
Used to detect HIV in suspected AIDS patients.
-
- It is used to detect mutations in genes in suspected cancer patients.
TRANSGENIC ANIMALS
Transgenic animals are animals who’s DNA possess a foreign
2. rDNA technology gene and express it.
(Step-1) Clone of cells are isolated from suspect E.g. Transgenic rats, rabbits, pigs, sheep, cows and fish.
(Step-2) A probe (ssDNA or ssRNA tagged with a radioactive
Reasons for developing transgenic animals:-
molecule) is allowed to hybridise to its complementary DNA (a) To study normal physiology & development:
in the cells Transgenic animals are used to study how genes are regulated, and
(Step-3) Cells are detected using autoradiography
how they affect the normal body functions and its development.
Case-I If normal DNA- Probe will hybridise with DNA and
radiation will appear on photographic film. (b) To study the contribution of genes in the development of a
Case-II If mutated DNA- Probe will not hybridise with DNA since it
disease and search for its treatment.
have no complimentarity with the mutated gene and hence (c) To obtain biological products:
the cells will not appear on the photographic film. E.g. 1. human protein (-1-antitrypsin) used to treat emphysema.
2. products for treatment of phenylketonuria (PKU) and
cystic fibrosis etc.
3. human protein-enriched milk
Rosie (first transgenic cow) produced milk containing the human
-lactalbumin and is nutritionally more balanced product for
human babies than natural cow-milk.
DNA
Rosie
(d) To test vaccine safety:
Transgenic mice are used to test the safety of the polio vaccine.
If it is found to be reliable, they can replace the use of monkeys to
test the safety of the vaccine.
(e) To test chemical safety:
Transgenic animals are made more sensitive to toxic substances.
It gives immediate results.
3. ELISA (Enzyme Linked Immuno-sorbent Assay)
Infection by pathogen can be detected by the presence of antigens
or by detecting the antibodies synthesized against the pathogen in a
GEAC & Biopiracy
sample. The current interest in the manipulation of microbes, plants, and
animals has raised serious ethical questions.
GEAC (Genetic Engineering Approval Committee) is an
organisation formed by Indian Government, to make decisions
about the validity and safety GM products.
Biopiracy: It is the use of bio-resources of developing countries
by multinational companies without proper authorization from the
countries and people concerned without compensatory payment.
E.g. American company patented Basmati rice which is actually
been derived from Indian variety, herbal medicines like turmeric
neem etc.
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