Genetically Modified crop-based Agriculture — It is responsible for Green Revolution
Characteristics of Genetic Modification in Crops
(i) Made crop more tolerant to abiotic stress (cold, drought, salt etc.)
(ii) Reduced reliance on chemical pesticides (pest resistant crop)
(iii) Helped to reduce post harvest losses (RNA interference)
(iv) Increased efficiency of mineral usage by plant (prevents early exhaustion of fertility of soil)
(v) Enhanced Nutritional Value of food (e.g. Golden Rice → Vitamin A)
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GMO — Genetically Modified Organisms
Section 02
GMO: Plants, Bacteria, Fungi and Animals whose genes have been altered by manipulation.
💡 Key point: Genetically Modified Organisms are created using Recombinant DNA Technology (rDNA). The foreign gene introduced is called Transgene, and the resulting organism is called Transgenic organism.
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Golden Rice & Flavr Savr Tomato
Section 03
Golden Rice (Oryza sativa)
Transgenic variety of rice
Contains β-Carotene (Pro-Vitamin A)
Process: Daffodil → Isolation of β-Carotene gene → Modified Ti Plasmid (using RE) → Recombinant → Transformation → Enter in template strain of rice → Golden Rice with Pro-Vitamin A
Flavr Savr Tomato
Transgenic Tomato
Gene responsible for softening of fruit (Enzyme: Polygalacturanase) is blocked using RNA interference / Antisense RNA / Complementary dsRNA
dsRNA blocks gene expression → enzyme not produced → fruit does not soften quickly → Longer shelf life
📌 Flavr Savr Tomato was the first genetically modified food approved for human consumption. RNA i blocks the gene for polygalacturanase enzyme which is responsible for softening of fruit during ripening.
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Bt Toxin & Bt Cotton
Section 04
Bt Toxin
Bt-toxin is produced by bacterium called Bacillus thuringiensis. In affected crop it creates Biopesticide. Examples: Bt Cotton, Bt Corn, Rice, Tomato, Potato & Soyabean.
Insect ingests it → Entry in midgut (Alkaline medium)
Activation of Endotoxin by Protease Enzyme
Activated toxin → Attachment with epithelium of midgut → Create Pores → Death of Insect
Bt Cotton — Insects it Controls
Order
Insects
Cry Gene
Lepidopterans
Corn Borer, Cotton Ballworm, Tobacco Budworm, Army Worm
Cry I Ab (Corn Borer) · Cry II Ab & Cry I Ac (Cotton Ballworm)
Coleopterans
Beetles
—
Dipterans
Flies & Mosquitoes
—
📌 Noxin (Bt protein) is coded by gene Cry I Ac named Cry. The protein encoded by gene controls specific insects. Cry I Ab → Corn Borer; Cry II Ab & Cry I Ac → Cotton Ballworm.
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RNA Interference (RNAi)
Section 05
What is RNA Interference?
RNAi takes place in all eukaryotic organisms as a method of cellular defence
Involves silencing of specific mRNA due to complementary dsRNA molecule that binds to mRNA and prevents translation (silencing)
Nematode Control using RNAi
Nematode Meloidogyne incognita infects the root of tobacco plants and causes great reduction in yield. Several nematodes parasite a wide variety of plants and animals including human beings.
Solution: Using Agrobacterium Ti Plasmid (after modification) → introduce nematode-specific gene into plant that produces both sense RNA and antisense RNA → forms dsRNA → RISC (RNA Inducing Silencing Complex) → Breakdown of sense RNA → Antisense RNA → Prevents protein formation → Stops parasitism
Step
Process
1. Nematode-specific gene
Introduced into plant via modified Ti Plasmid
2. Both Sense + Antisense RNA produced
Complementary strands pair up → dsRNA formed
3. RISC activation
RNA Inducing Silencing Complex processes dsRNA
4. Sense mRNA breakdown
RISC with Antisense RNA destroys specific mRNA
5. Result
Nematode protein not formed → Nematode dies → No parasitism
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Biotechnology in Medicine — Genetically Engineered Insulin
Section 06
At present 30 recombinant therapeutics have been approved for human use worldwide. In India, twelve of these are presently being marketed.
Structure of Insulin
Consists of two short polypeptide chains: Chain A and Chain B linked together by Disulphide Bond
In mammals, insulin acts as Prohormone in which extra chain called C-Peptide is present
A chain → 21 amino acids · B chain → 30 amino acids · C chain → 33 amino acids
Total in mature insulin → 51 amino acids
Maturation: Proinsulin → Insulin by removal of C-Peptide & addition of A and B chain by disulphide bond
Production of Humulin (Human Insulin)
In 1983, Eli Lilly (American Company) introduced A and B chains of human insulin into E. coli to produce insulin chains
A & B genes isolated from Chromosome 11 of human cell
Chains produced separately and joined by Disulphide bond to form human insulin
Final product called Mature Insulin (Humulin)
📌 Key facts: A chain = 21 amino acids · B chain = 30 amino acids · C chain = 33 amino acids (removed during maturation) · Total in mature insulin = 51 amino acids · Gene on Chromosome 11.
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Gene Therapy
Section 07
What is Gene Therapy?
Correction of a gene defect in a patient by delivery of a healthy gene. The first clinical gene therapy was given in 1990 to a 4-year-old girl with Adenosine Deaminase (ADA) deficiency.
ADA Deficiency (SCID)
Caused due to deletion of Adenosine Deaminase (ADA) gene
ADA deficiency can be cured by: Bone marrow transplantation & Enzyme replacement therapy (ADA injection) — but not completely curative
Gene therapy: If gene isolated from bone marrow cells producing ADA is introduced into cells at embryonic stage — could be a permanent cure
Gene Therapy Process for ADA Deficiency
Patient (SCID → ADA Deficiency)
Isolation of ADA defective cells from patient
Healthy ADA gene introduced into Retrovirus (Vector)
Retrovirus (with ADA gene) introduced into patient cells
ADA gene → produces ADA enzyme (protein) → Stimulates proliferation of T-Lymphocytes (CMI) and B-Lymphocytes (AMI)
Healthy cells returned to patient → Growth in culture → Healthy cells with Healthy ADA gene
⚠️ Gene therapy provides a functional copy of the gene but not a permanent cure unless introduced at embryonic stage. The therapy must be repeated periodically as the T-lymphocytes produced are not immortal.
Recombinant DNA Technology, PCR, and Enzyme Linked Immuno Sorbent Assay (ELISA) are some techniques used for early diagnosis.
Autoradiography (PCR-based Detection)
A ssDNA or RNA tagged with Probe (radioactive molecule — P³²) is allowed to hybridize to its complementary DNA in a clone of cells followed by detection using Autoradiography. Clone having mutated gene will NOT appear on photographic film because Probe will not have complementary with mutated gene.
Process: Collection of blood sample → Isolation of DNA/RNA → Amplification of DNA by PCR → Gel Electrophoresis → Hybridization (with Probe/ssDNA-RNA/P³²-radioactive)
ELISA (Enzyme Linked Immuno Sorbent Assay)
ELISA is based on Antigen-Antibody interaction. Infection by pathogen can be detected by presence of antigens (proteins, glycoproteins) or by detecting the antibodies synthesised against pathogen.
Symbol
Represents
●
Antigen
Y (λ)
Antibody
Y with enzyme (λᴱ)
Enzyme (Peroxidase) Linked Antibody
S
Substrate
P
Product (colour change — positive test)
💡 ELISA uses enzyme-linked antibodies. If antigen is present, antibody binds → enzyme-linked antibody binds → substrate added → colour change = Positive test. Used for detecting HIV, hepatitis B, pregnancy tests, etc.
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Transgenic Animals
Section 09
Transgenic Animals: Animals that have their DNA manipulated to possess and express a foreign gene. 95% of all transgenic animals are mice.
Benefits of Transgenic Animals
Benefit
Details
(i) Normal Physiology & Development
Transgenic animals designed to study how genes are regulated and how they affect normal body functions
(ii) Study of Disease
Transgenic models exist for many human diseases such as Cancer, Cystic Fibrosis, Rheumatoid Arthritis, Alzheimer's
(iii) Biological Products
Human protein α-1 anti-trypsin used to treat emphysema. In 1997, first transgenic cow Rosie produced human protein enriched milk (2.4 gm/litre) containing human alpha-lactalbumin — nutritionally more balanced for human babies than natural cow milk
(iv) Vaccine Safety
Transgenic mice used to test safety of Polio vaccine
(v) Chemical Safety Testing
Toxicity testing in transgenic animals gives results in less time
⚖️
Ethical Issues, Biopiracy & Patents
Section 10
Ethical Issues — GEAC
Indian Government set up organisation GEAC (Genetic Engineering Approval Committee) which makes decisions regarding GM research and safety of introducing GMOs for public services.
Patent
A set of exclusive rights granted by national government to an inventor.
Criteria of Patent: (i) Novelty · (ii) Non-Obviousness · (iii) Utility
Biopiracy
Use of bioresources by multinational companies and other organisations without proper authorisation from the countries and people concerned, without compensatory payment.
Examples of Biopiracy
Basmati Rice: There are estimated 2,00,000 varieties of rice in India. Basmati is distinct for its aroma & flavour — 27 documented varieties grown in India. In 1997, an American company got patent rights on Basmati rice by US Patent & Trademark office. New variety of Basmati was actually derived by Indian farmers and crossed with semi-dwarf varieties — claimed as invention or novelty.
Herbal Medicine: Several attempts made to get patent on herbal medicine e.g. Turmeric and Neem.
Pentadiplandra Brazzaana (West African plant) — produces a protein called Brazzein (2000 times sweeter than normal sugar) — patented by USA.
⚠️ Indian Parliament recently cleared the Second Amendment of Indian Patent Bills that takes such issues into consideration including patent terms, emergency provision and research and development initiative. This protects traditional knowledge from being exploited.
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MCQ Practice — Biotechnology Applications
Section 11
Q1. Bt toxin is activated in the midgut of insects because the midgut is:
Bt toxin (Cry protein) is inactive when produced. When ingested by insect, it enters the alkaline midgut where Protease enzymes activate it. The activated toxin attaches to epithelium, creates pores and causes death of insect.
Q2. The first clinical gene therapy was given in 1990 for:
The first clinical gene therapy was given in 1990 to a 4-year-old girl with Adenosine Deaminase (ADA) deficiency, which causes Severe Combined Immune Deficiency (SCID). ADA deficiency is caused due to deletion of the ADA gene.
Q3. Flavr Savr tomato has longer shelf life because:
Flavr Savr is a transgenic tomato where the gene responsible for softening of fruit (Enzyme: Polygalacturanase) is blocked using RNA interference / Antisense RNA / Complementary dsRNA. The dsRNA blocks gene expression so the enzyme is not produced and fruit does not soften quickly.
Q4. Golden Rice is rich in:
Golden Rice (Oryza sativa) is a transgenic variety that contains β-Carotene (Pro-Vitamin A). The β-Carotene gene was isolated from Daffodil and introduced into rice using Modified Ti Plasmid (from Agrobacterium).
Q5. Humulin (genetically engineered insulin) was first introduced by which company in 1983?
In 1983, Eli Lilly, an American company, introduced A and B chains of human insulin into E. coli separately. They were produced separately and joined by disulphide bonds to form human insulin (Humulin). A & B genes were isolated from Chromosome 11.
Q6. Nematode Meloidogyne incognita infects the roots of which plant causing reduction in yield?
Nematode Meloidogyne incognita infects the root of tobacco plants and causes great reduction in yield. This is controlled using RNA interference — nematode-specific dsRNA is introduced into plant via Agrobacterium Ti Plasmid, silencing the nematode's survival gene.
❓ Frequently Asked Questions
Bt toxin is produced by Bacillus thuringiensis. It is a protein (Cry protein) encoded by cry genes. When insects ingest it, the inactive endotoxin enters the alkaline midgut and gets activated by protease enzymes. Activated toxin attaches to epithelium of midgut, creates pores and causes death of insect. Different Cry genes control different insects: Cry I Ab → Corn Borer; Cry I Ac → Cotton Bollworm.
RNA interference is a method of cellular defence in all eukaryotic organisms. It involves silencing of specific mRNA by complementary dsRNA that binds to mRNA and prevents translation. RISC (RNA Inducing Silencing Complex) processes the dsRNA. Used to create nematode-resistant tobacco plants by introducing nematode-specific dsRNA via Agrobacterium Ti Plasmid.
In 1983, Eli Lilly introduced A and B chain genes (from Chromosome 11) separately into E. coli. A chain (21 amino acids) and B chain (30 amino acids) were produced separately and joined by disulphide bonds to form mature human insulin (Humulin). C-peptide (33 amino acids) is removed during conversion of proinsulin to insulin.
The first clinical gene therapy was given in 1990 to a 4-year-old girl with ADA deficiency (caused by deletion of adenosine deaminase gene). Healthy ADA gene was isolated, introduced into Retrovirus (vector), which was used to infect patient's cells. Healthy cells with ADA gene were returned to patient to stimulate immune system.
Biopiracy is the use of bioresources by multinational companies without proper authorisation or compensatory payment. Examples: (1) In 1997, an American company patented Basmati rice using US Patent & Trademark office, despite being developed by Indian farmers. (2) Attempts to patent Turmeric and Neem. (3) Pentadiplandra Brazzaana (West African plant producing Brazzein — 2000x sweeter than sugar) patented by USA.
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